Phenotype-Dependent Expression of α-Smooth Muscle Actin in Visceral Smooth Muscle Cells

Experimental Cell Research 247, 279 –292 (1999) Article ID excr.1998.4339, available online at http://www.idealibrary.com on

Phenotype-Dependent Expression of a-Smooth Muscle Actin in Visceral Smooth Muscle Cells Hiroshi Saga, 1 Kazuhiro Kimura, 1 Ken’ichiro Hayashi, Takahiro Gotow,* Yasuo Uchiyama,* Takuya Momiyama, Satoko Tadokoro, Nozomu Kawashima, Akie Jimbou, and Kenji Sobue 2 Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, and *Department of Anatomy, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

a-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, a-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (2238 to 2219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of a-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors. © 1999 Academic Press

Key Words: a-smooth muscle actin; smooth muscle cell; phenotypic modulation; transcription; negative element.

INTRODUCTION

The actin isoforms are divided into two classes, muscle and nonmuscle actins, and are designated accord1

These authors contributed equally to this work. To whom all correspondence and reprint requests should be addressed. Fax: 81-6-879-3689. E-mail: [email protected]. ac.jp (Kenji Sobue). 2

ing to their isoelectric points as a, b, and g. Of these, a-smooth muscle (a-SM) actin comprises a major portion of actin isoforms expressed in vascular SMCs [1, 2]. Expression of actin isoforms in rat and human aortic SMCs is developmentally regulated; b-nonmuscle actin is substituted by a-SM actin [3, 4]. Steady-state levels of a-SM actin mRNA increase in developing aortas, reaching about 90% of total actin mRNAs in adult tissue [5], whereas the expression of a-SM actin decreases in primarily cultured aortic SMCs in association with their phenotypic modulation [5, 6]. a-SM actin has been, therefore, used as a favorable molecular marker of vascular SMC phenotype. On the other hand, the presence of a-SM actin in visceral SMCs is complicated because of the following findings. Izant and Lazarides [7] reported a trace amount of a-actin coexisting with b- and g-actins in chicken gizzards from embryo to adult, and Saborio et al. [8] did not detect a-SM actin in 8- and 12-day-old embryo gizzards, but they found a trace of this protein in 17and 20-day-old embryos. These discrepant results were based on the identification of a-SM actin by two-dimensional gel electrophoresis. Immunofluorescence microscopy of a-SM actin in adult chicken gizzards revealed intensely positive staining in blood vessels, but not in parenchymal cells [2]. In addition to these findings, a-SM actin was ectopically detected in nonmuscle stromal cells [9]. Under certain pathological conditions, the enhanced expression of a-SM actin was also observed in massively proliferated mesangial cells [10] and myofibroblasts [11]. In the present study, we clearly demonstrated the expression of a-SM actin in undifferentiated and dedifferentiated visceral SMCs, but not in differentiated visceral SMCs. This is the first report regarding the opposite expression of a-SM actin in vascular and visceral SMCs in their phenotype-dependent manner. We also investigated the transcriptional regulation of this gene in both differentiated and dedifferentiated phenotypes of visceral SMCs and identified a novel negative element in the a-SM actin promoter.

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MATERIALS AND METHODS Immunocytochemistry. Gizzards, small intestines, tracheas, and aortas at indicated developmental stages were excised, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and cryoprotected by 30% sucrose in phosphate-buffered saline (PBS). Thin sections of each tissue were stacked on gelatine-coated slide glasses and stained with anti-a-SM actin monoclonal antibody (anti-a-sm-1, Sigma) using fluorescein isothiocyanate (FITC)-labeled anti-mouse antibody, and directly with rhodamine-phalloidin for total F-actin. The specimens were viewed with a Zeiss LSM 410 confocal microscope (Carl Zeiss). Cell culture. Gizzard SMCs for primary culture were prepared from 15-day-old chick embryos as described elsewhere [12, 13]. Gizzard SMCs in a differentiated phenotype were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 0.2% BSA and 5 mg/ml insulin on laminin-coated 12- or 6-well plates (5 3 10 5 cells/3.6 cm 2 well or 1–1.5 3 10 6 cells/9.2 cm 2 well). Under these conditions, differentiated SMCs were cultured for more than 1 week. To promote dedifferentiation, the isolated gizzard SMCs were cultured in DMEM supplemented with 10% fetal calf serum on noncoated plastic culture plates for more than 1 week. Two-dimensional electrophoresis. Two-dimensional gel electrophoresis was performed as described by Hirai and Hirabayashi [14] with some modifications. The tissues were homogenized with 9.5 M urea solution, and the urea extracts were separated by two-dimensional gel electrophoresis. The spots of each actin isoform stained by Coomassie brilliant blue were quantified by densitometric scanning with Molecular Dynamics (American Megtrends Inc.). The separated actin isoforms were also probed with anti-a-SM actin antibody. Immunoblotting. After washing with PBS, the cells were lysed with 2% SDS sample buffer. The protein samples were separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Detection of target proteins on the membrane was performed by an ECL Western blotting detection kit (Amersham) using respective antibodies. Anti-g-SM actin monoclonal antibody was purchased from ICN. Anti-caldesmon and anti-vinculin polyclonal antibodies were prepared [15, 16], and anti-calponin polyclonal antibody was donated by Dr. K. Hiwada [17]. Northern blotting. Total cellular RNAs (2 mg) from gizzards or cultured gizzard SMCs under indicated conditions were separated by 1% formaldehyde agarose gels and transferred to a nylon membrane. Antisense oligonucleotide corresponding to nucleotide positions from 12312 to 12331 in a-SM actin cDNA [18] was used as a probe. The membrane was hybridized at 55°C with the end-labeled probe with [g- 32P]ATP and was washed three times with 6X SSC, 0.1% SDS at 55°C for 30 min. To visualize rRNAs, the membranes were stained with 0.02% methylene blue. Hybridization with the indicated probes, caldesmon, a-tropomyosin, calponin, b-tropomyosin, and a1 integrin, was carried out as described elsewhere [12, 13]. Promoter analysis. A chicken genomic library constructed in l DASH II was screened using chicken a-SM actin cDNA prepared by reverse transcription-polymerase chain reaction (RT-PCR) as a probe. Genomic clones carrying the 59-upstream region of the a-SM actin gene were characterized by Southern blotting, and the 59upstream region (2984 to 140) was amplified by PCR. An amplified 1030-bp fragment was sequenced to confirm its accuracy and then was inserted into the SmaI site of pUC0CAT, promoter-less chloramphenicol acetyltransferase (CAT) plasmid [19]. This plasmid carrying the CAT reporter gene under the 59-upstream sequence of a-SM actin gene was designated as pActCAT984. Deletions and/or mutations derived from pActCAT984 were prepared using restriction sites (HindIII, PstI, and SacI) and PCR methods. Transfections and CAT assays [20] were carried out as follows. Calcium phosphateDNA precipitates containing 2 mg of CAT construct plus 1 mg of

control plasmid carrying the luciferase cDNA under Rous sarcoma virus promoter (pRSV-luciferase) were added to cultured gizzard SMCs as described elsewhere [21]. In the case of forced expression of MSSP-1, 50 ng of pEF-MSSP-1 [22, 23] was cotransfected with CAT construct and pRSV-luciferase. Standardization of transfection efficiency was carried out using luciferase activity [24]. The appropriate volume of the cell extracts after heating to inactivate endogenous deacetylases was incubated at 37°C with 1 mM acetyl-coenzyme A and 3.7 kBq of [ 14C]chloramphenicol (Amersham) and analyzed by thin-layer chromatography. pUC0CAT and pUC2CAT carrying the CAT gene under the SV40 early promoter and enhancer [19] were used as negative and positive controls, respectively. The transfection experiments were repeated on multiple sets of cultures with two or three different plasmid preparations. CAT activities were quantified by Scanning Imager (Molecular Dynamics) and were normalized to the activity of pUC2CAT in respective cells as 100%. We confirmed that transfection efficiency was constantly similar in both differentiated and dedifferentiated SMCs as judged by active staining of b-galactosidase activity from a reporter plasmid carrying a b-galactosidase gene downstream from the SV40 early promoter using X-gal as a substrate. The activity of pUC2CAT was not significantly affected under SMC culture conditions; the CAT activities were identical in SMCs cultured under serum-stimulated or serum-free conditions (data not shown). Under serum-free culture conditions, caldesmon promoter/CAT constructs showed high levels of promoter activity [21]. Analysis of DNA-protein interaction by gel-shift assay. For characterization of DNA-protein interaction, samples of nuclear extracts (4 mg) were mixed with 0.1– 0.2 ng of 32P-labeled probe and 2 mg of poly(dI-dC) in the presence or absence of unlabeled competitor at room temperature for 20 min in 20 ml containing 5 mM N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid (Hepes), pH 7.8, 5 mM 2-mercaptoethanol, 1 mM EDTA, 60 mM NaCl, 5 mM spermidine, and 10% glycerol. Samples for gel-shift assay were analyzed on 7% polyacrylamide gels in 0.5X TBE buffer. Anti-serum response factor (SRF) polyclonal antibodies were purchased from Santa Cruz Biotechnology. We confirmed the cross-reactivity of these antibodies to chicken SRF [25].

RESULTS

Expression of a-SM Actin in Different Smooth Muscle Tissues during Development We investigated the expression of chicken a-SM actin in different smooth muscle tissues at the indicated developmental stages (Fig. 1). In this study, we used anti-a-sm-1 as a-SM actin-specific antibody [2] which has been demonstrated to recognize the amino-terminal sequence of a-SM actin, AcEEED [26]. This sequence is conserved in chicken and mammalian a-SM actins. The smooth muscle layers in visceral tissues (indicated by SM) including gizzard, intestine, and trachea in a 10-day-old embryo (a, d, and g) were labeled with both a-SM actin antibody (green) and phalloidin (red). The staining with the antibody was more intense, indicating the high expression of a-SM actin in these muscle layers. The a-SM actin staining dramatically decreased in the smooth muscle layers of a 15day-old embryo (b, e, and h) and was less significant at 1 month after hatching (c, f, and i). Curiously, in the small intestine of a 15-day-old embryo, the inner layer

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FIG. 1. Expression of a-SM actin in gizzard, small intestine, trachea, and aorta at different developmental stages. The smooth muscle layers in visceral and vascular tissues were stained with a-SM actin antibody (green) and rhodamine-phalloidin (red): gizzard (a, b, and c); small intestine (d, e, and f); trachea (g, h, and i); and aorta (j, k, and l). The developmental stages are as follows: 10-day-old embryo (a, d, g, and j); 15-day-old embryo (b, e, h, and k); and 1-month-old chicken after hatching (c, f, i, and l). Arrows in c and i indicate the a-SM actin staining of small blood vessels within the visceral tissues. Arrowheads in e and f are the inner layer of intestinal smooth muscle layers. Bar, 100 mm.

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of smooth muscle layers retained the a-SM actin staining, which was observed until 1 month old (e and f, indicated by arrowheads). In aortic media, the a-SM actin staining was not conspicuous in 10-day-old embryo (j), but began to increase in a 15-day-old embryo (k) and thereafter (l). Developmental changes of a-SM actin staining were also seen in blood vessels supplied to visceral tissues (indicated by arrows in c and i). The biochemical detection of a-SM actin protein with immunoblotting in developing gizzard and aortic muscles (from 10-day-old embryos to 1 month after hatching) coincided well with the immunocytochemistry as described above (Fig. 2A). We further quantified the expression of a-SM actin protein by two-dimensional gel electrophoresis (Fig. 2B). The a-actin expression in gizzard was found in 10-day-old embryos (15.8% of total actin contents) and 15-day-old embryos (2.0% of total actin contents) and was not detected thereafter. The relative ratio of b-actin also decreased in developing gizzard (34.8 to 14.6%), whereas that of g-actin increased (49.4 to 85.4%). In developing aortic muscles, a-SM actin was upregulated, whereas b-actin was downregulated and g-actin remained at nearly equal levels. The relative ratios of each actin in total actin isoforms during aortic development were as follows: 18.7 to 56.1% (a-actin), 46.1 to 15.7% (b-actin), and 35.1 to 28.1% (g-actin). These results are consistent with those in developing rat aortic media reported by Owens and Thompson [3]. Expression Change of a-SM Actin during Phenotypic Modulation of SMCs It is well known that cultured SMCs stimulated by serum rapidly convert their phenotype from a differentiated to a dedifferentiated state [27]. During this process, expression changes of cytoskeletal and contractile proteins are also observed. For example, the a-SM actin expression is downregulated during dedifferentiation of aortic SMCs [5, 6]. Our results in Figs. 1 and 2 suggest that the expression patterns of a-SM actin during visceral SMC dedifferentiation and development would be a mirror image. To analyze the molecular events during phenotypic modulation of visceral SMCs, we established an SMC culture system maintaining a differentiated phenotype. To our knowledge, there have been no reports regarding clonal cell lines of SMC that can control their own phenotype. Further, primarily cultured SMCs under conventional conditions rapidly change their phenotype from a differentiated to a dedifferentiated state and passaged SMCs cannot retrieve a differentiated phenotype even if they are cultured under quiescent conditions. Extracellular matrices (ECMs) have been reported to be involved in such phenotypic modulation [28]. We compared the

effects of ECM components on a phenotype of SMC. Among laminin, fibronectin, and collagens type-I and type-IV examined, laminin had a relative potency to retard SMC dedifferentiation as monitored by isoform conversion of caldesmon protein (Fig. 3A). Laminin was, however, not able to maintain a differentiated phenotype of SMCs for a long culture, suggesting that additional factor would be required. We then examined several growth factors and cytokines on a phenotype of SMCs cultured on laminin. Dedifferentiation of SMCs was induced by serum and several growth factors/cytokines, whereas insulin was able to prompt SMCs to maintain a differentiated phenotype for more than 7 days as determined by the expression of SMC-specific molecular markers such as h-caldesmon, SMC-specific a-tropomyosin, calponin, b-tropomyosin, and a1 integrin at mRNA levels (Fig. 3B). By contrast, in SMCs cultured under serum-stimulated conditions, h-caldesmon mRNA and SMC-specific a-tropomyosin mRNA were converted to l-caldesmon mRNA and fibroblasttype a-tropomyosin mRNA carrying the exon 2b, respectively. Further, in the cells, calponin, b-tropomyosin, and a1 integrin mRNAs were downregulated to undetectable levels. Besides the molecular markers, SMCs cultured under the above conditions showed a spindle-like shape, formed a typical meshwork, and possessed a carbachol-induced contractility (Fig. 3C, a and c). On the other hand, SMCs cultured under serum-stimulated conditions showed a fibroblast-like shape and were unable to contract (Fig. 3C, b and d). We used the present culture system to analyze the phenotype-dependent expression of a-SM actin in visceral SMCs. When SMCs were cultured on laminin under insulinstimulated conditions, cell growth was arrested (Fig. 4A). The SMCs did not express a-SM actin even in 7-day cultures, but they potently expressed SMC-specific marker proteins, such as h-caldesmon, calponin, meta-vinculin, and g-SM actin for more than 7 days (Fig. 4B). During serum-induced dedifferentiation, hcaldesmon converted to l-caldesmon, and total caldesmons, calponin, meta-vinculin, and g-SM actin were downregulated (Fig. 5B). By contrast, the a-SM actin was detected at 2 days of culture and dramatically increased thereafter, and this expression remained constant even in a postconfluent state (Fig. 5B). Isoform conversion of caldesmon and downregulation of calponin, meta-vinculin, and a-SM actin began to occur at 2 days of culture (Fig. 5B). A change of actin isoforms in gizzard SMCs was also analyzed by two-dimensional gel electrophoresis (data not shown). Between 0- and 7-day cultures, the relative ratio of a-actin in total actin isoforms increased from 2.8 to 19.0%. b-Actin also increased from 28.1 to 50.6%, but g-actin decreased from 69.0 to 30.3%.

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FIG. 2. Expression change of actin isoforms during development of chicken gizzard and aorta smooth muscles. (A) Tissue extracts from developing gizzard and aortic muscles (10-day-old (E10), 15-day-old embryos (E15), and 1 month after hatching (1M)) were separated by SDS-polyacrylamide gel electrophoresis and the expression of a-SM actin was probed with anti-a-SM actin antibody. (B) The tissue extracts from chicken gizzard and aortic smooth muscles of 10-day-old (a, d), 15-day-old (b, e) chick embryos, and 1-month-old chicken after hatching (c, f) were separated by two-dimensional gel electrophoresis. The actins were stained with Coomassie blue (a, b, c) and a-SM actin was identified by immunoblotting with anti-a-SM actin antibody (d, e, f). The immunoreacting spots corresponded to the polypeptides migrating to the positions of a-actin (arrowheads).

We then investigated the expression of a-SM actin mRNA by Northern blotting (Fig. 5C). Four kinds of mRNAs (1.3–2.7 kb) were hybridized with the a-SM actin-specific probe. A difference in mRNA size depends on the selection of four polyadenylation signals located in the 39-downstream region of this gene [29].

While the a-SM actin mRNAs were not expressed before culture, they were expressed at 1 day of culture and increased thereafter (Fig. 5C). The expression of a-SM actin protein and mRNAs correlated with each other and did not correlate with cell proliferation. Taken together, the present results suggest that a phe-

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FIG. 3. Primary culture system of gizzard SMCs maintaining a differentiated state. (A) Effects of ECM components on the expression of caldesmon isoforms (h-caldesmon (h-CaD) and l-caldesmon (l-CaD)) in cultured SMCs under serum-free conditions. Gizzard SMCs from 15-day-old chicken embryos were cultured under indicated conditions and whole cell lysates were analyzed by immunoblotting with antibodies against caldesmon. Anti-caldesmon antibodies reacted with both h- and l-caldesmons. ECM components and culture days were indicated at the top: N, noncoated plate; LN, laminin; FN, fibronectin; CI, collagen type-I; CIV, collagen type-IV; and P, precultured SMCs. (B) Northern blotting showed the expression of SMC-specific molecular marker genes in 7-day culture of SMCs on laminin under insulin-stimulated conditions (I) and on noncoated plate under serum-stimulated conditions (S). Probes used for hybridization were indicated at bottoms, caldesmon (CaD), a-tropomyosin (a-TM), calponin (CN), b-tropomyosin (b-TM), and a1 integrin. The caldesmon probe specifically hybridized to both h-caldesmon mRNA (h-CaD, 4.8 kb) and l-caldesmon mRNA (l-CaD, 4.1 kb). Two a-TM probes, E2a and E2b, are specific to exons 2a and 2b in the a-tropomyosin gene, respectively. The exon 2a is specifically spliced in SMC-specific a-tropomyosin mRNA, whereas the exon 2b is spliced in several a-tropomyosin mRNAs which are expressed in dedifferentiated SMCs and other cells except for SMCs [12]. mRNAs hybridized to indicated probes are shown in the top and middle panels, and 28S rRNAs stained by methylene blue are shown in the bottom panels. (C) Morphology and ligand-induced contractility of cultured SMCs. SMCs were cultured on laminin under insulin-stimulated conditions (a and c) and on noncoated plates under serum-stimulated conditions (b and d), and contraction was induced by an addition of carbachol (1 mM) for 1 min. Photographs are shown as before (a and b) and after (c and d) carbachol treatment.

notype-dependent change of a-SM actin expression in gizzard SMCs is regulated at a transcriptional level. Identification of Visceral SMC Phenotype-Dependent Cis-Elements in the a-SM Actin Promoter To investigate the transcriptional regulation of the a-SM actin gene, we analyzed the a-SM actin promoter in both differentiated and dedifferentiated gizzard SMCs. Figure 6A shows a schematic diagram of the 59-upstream region of the chicken a-SM actin gene from 2984 to 140 [29] and a series of deleted CAT constructs. The promoter activities of CAT constructs in both phenotypes of SMCs are shown in Figs. 6B and 6C. The promoter activities of the constructs from

pActCAT984 to pActCAT238 were negligible in differentiated SMCs, whereas these constructs produced potent activities in dedifferentiated SMCs. These results are in accordance with immunoblotting and Northern blotting of endogenous expression of a-SM actin (Fig. 4B and Figs. 5B and 5C). pActCAT193 and pActCAT137 in differentiated SMCs produced significant promoter activities, whereas pActCAT106 did not (Fig. 6B). In dedifferentiated SMCs, pActCAT193 also produced high levels of promoter activity. These results suggest that a negative element is located within a sequence expanding from 2238 to 2194. To identify the negative element, we analyzed promoter activities of several deleted and/or mutated constructs. A se-

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quence expanding from 2219 to 2214, CAGCTG, was the consensus sequence of E box, while the effects of E box deletion or mutation on the promoter activity were less significant (data not shown). From these findings, the negative element is considered to be restricted within a sequence expanding from 2238 to 2219. In fact, mutation within this region resulted in enhancement of the promoter activity in both differentiated and dedifferentiated SMCs (Fig. 7). These results suggest that a sequence from 2238 to 2219 plays a role for a negative element. The promoter activities of pActCAT193, pAct137CAT, and pAct106CAT gradually decreased in dedifferentiated SMCs (Fig. 6C), indicating that the positive elements are located within two different regions, expanding from 2193 to 2138 and from 2137 to 2107. Positive regulatory elements of the a-SM actin promoter in some species have been well characterized. A purinerich motif (GGAATG) expanding from 2181 to 2176 and two CArG box-like motifs, CArG B (CCCTATATGG) expanding from 2120 to 2111 and CArG A (CCTT-

FIG. 5. Expression change of a-SM actin during dedifferentiation process of cultured gizzard SMCs. Gizzard SMCs from 15-dayold chick embryos were cultured on noncoated plates under serumstimulated conditions. Progressive changes in cell numbers (A) and expression changes of a-SM actin and other SMC-specific molecular marker proteins (B) are shown as Fig. 4. Expression change of a-SM actin mRNAs is shown (C).

FIG. 4. No expression of a-SM actin in cultured gizzard SMCs maintaining a differentiated state. Gizzard SMCs from 15-day-old chick embryos were cultured on laminin under insulin-stimulated conditions. Progressive change in cell numbers under these culture conditions is shown (A). Expression changes of a-SM actin and other SMC-specific molecular marker proteins were probed by antibodies against a-SM actin, caldesmon (CaD), calponin (CN), vinculin (VIN), and g-SM actin. Anti-vinculin antibodies reacted with both vinculin and meta-vinculin (meta-VIN).

GTTTGG) expanding from 270 to 261, function as positive elements in cultured rat aortic SMCs, mouse AKR-2B embryonic fibroblasts, and BC3H1 myoblasts [30, 31]. The promoter analyses using deleted constructs showed that in differentiated gizzard SMCs, the CArG B functioned as a positive element but the purine-rich motif did not, whereas in dedifferentiated SMCs, both the CArG B and the purine-rich motif functioned as positive elements. To clarify the positive elements in a-SM actin promoter in both phenotypes of gizzard SMCs, we introduced mutations into the purine-rich motif, the CArG B or the CArG A in pAct-

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FIG. 6. Promoter analyses of the a-SM actin gene using a series of deleted CAT constructs in differentiated and dedifferentiated gizzard SMCs. The alignment map of the 59-upstream region of chicken a-SM actin gene (2984 to 140) and schematic structures of deleted CAT constructs are shown (A). Restriction sites, locations of canonical cis-elements, and the starting site of transcription are indicated in the map. E, Pu, B, A, and T mean the E box, the purine-rich motif, the CArG B, the CArG A, and TATA box, respectively. Results of CAT assays in both states of gizzard SMCs are graphically presented: differentiated state (B) and dedifferentiated state (C). CAT activities were normalized to the activity of pUC2CAT in respective cells as 100%.

CAT193 (Fig. 7A). In differentiated SMCs, mutation in the CArG B resulted in an 80% decrease in the promoter activity, while mutation in the purine-rich motif or the CArG A did not show such a drastic decrease (40 and 20% decreases) (Fig. 7B). In dedifferentiated SMCs, mutation in the purine-rich motif, the CArG B, or the CArG A led to an 88 to 93% decrease in the promoter activities (Fig. 7B). These results suggest that the CArG B acts solely as a positive element in differentiated SMCs, while the purine-rich motif and CArGs A and B cooperatively act as positive elements in dedifferentiated SMCs. Transacting Factors Interacting with the Negative and Positive Cis-Elements We further characterized the transacting factors to these cis-elements which were involved in visceral SMC phenotype-dependent transcription of a-SM actin genes. Regarding the negative element, we performed gel-shift assays to detect specific DNA-protein interactions. In this assay, we used NE20, a 20-bp DNA fragment expanding from 2238 to 2219, as a probe (Fig. 7A). Both nuclear extracts from differentiated and dedifferentiated SMCs formed a DNA-protein complex. The complex formation was suppressed by unlabeled NE20, but not by nonspecific DNA fragments (Fig. 8A). The nuclear extracts from dedifferentiated SMCs formed a more intense complex

than those from differentiated SMCs. These results suggest that specific DNA-protein interactions occur within the sequence of NE20. Since the gel-shifted positions of the complexes with NE20 and differentiated or dedifferentiated SMC nuclear extracts were identical, the same protein factors might be involved in the negative regulation of the a-SM actin gene. We have recently identified one of the protein factors interacting NE20 as a c-myc gene single-strand-binding protein-1 (MSSP-1) by Southwestern screening. Anti-MSSP-1 antibodies inhibited such complex formation with NE20 [32]. Forced expression of MSSP-1 in dedifferentiated SMCs resulted in a 50% decrease in the promoter activity of pActCAT238, whereas the activity of pActCAT238 (NEmut) was not affected (Fig. 8B). It has been reported that SRF binds to the CArGs A and B in rat a-SM actin promoter [31] and transcriptional enhancer factor 1 (TEF-1) binds to the purinerich motif in mouse a-SM actin promoter [30]. We further compared the involvement of these transacting factors in the transcriptional regulation of a-SM actin gene in both differentiated and dedifferentiated gizzard SMCs. Using anti-SRF antibodies, SRF was identified as one of the protein factors bound to the CArGs A and B (Fig. 9A). The CArG-SRF complex with nuclear extracts from differentiated SMCs was prominent, while that with dedifferentiated SMC nuclear

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FIG. 7. Effects of mutation in the negative element, the purine-rich motif, or the two CArG box-like motifs on the activities of the a-SM actin promoter. Mutations in the sequences of the negative element, the purine-rich motif, and the CArGs A and B are indicated by negative scripts (A). Mutations in the negative element were introduced in pActCAT238 and this construct was designated pActCAT238(NEmut). Mutations in the purine-rich motif, the CArG B, or the CArG A were introduced in pActCAT193. Respective mutated constructs were designated pActCAT193(Pumut), pActCAT193(Bmut), and pActCAT193(Amut). Relative effects of mutations in both differentiated (open boxes) and dedifferentiated SMCs (closed boxes) are shown (B). In this case, promoter activities were normalized to the activity of pActCAT193 in both phenotypes of SMCs as 100%.

extracts was faint, and the complex formation with the CArG B was stronger than that with the CArG A (Fig. 9A). We also confirmed the CArG A-SRF complex formation with dedifferentiated SMC nuclear extracts by gel-shift assays using a highly radioactive probe (data not shown). Similar results were also observed in the promoter analyses of a1 integrin [13] and caldesmon genes [25]; the affinity of SRF in the nuclear extracts from differentiated SMCs to the CArG box-like motif is much higher than that from dedifferentiated SMCs. We also characterized the expression of SRF mRNA during serum-induced dedifferentiation of SMCs [13, 25]. The expression of endogenous SRF in both differentiated and dedifferentiated SMCs did not drastically change as observed in gel-shift assays; the SRF mRNA in dedifferentiated SMCs was twofold lower than that in differentiated SMCs. These results suggest that additional factor(s) would modulate the activity of SRF to enhance the transcription. The specific DNA-protein complex was detected using Pu23, a 23-bp DNA fragment containing the pu-

rine-rich motif as a probe (Figs. 7A and 9C). Both nuclear extracts from differentiated and dedifferentiated SMCs formed an identical DNA-protein complex (Fig. 9C), and the protein factor in SMC nuclear extracts was identified as TEF-1 by competition assay using an oligonucleotide containing TEF-1-binding sequence, GCATGCTTTGCATACTT (data not shown). DISCUSSION

a-SM actin has been well characterized as a molecular marker for vascular SMC phenotypes. However, this actin isoform undergoes ectopic expression in embryonic striated muscles [33] and certain stromal cells [9]. As described in the Introduction, the evaluation of this isoform in visceral SMCs has been conflicting. Here, we have first demonstrated the phenotype-dependent expression of a-SM actin in visceral SMCs, which is a mirror image of that in vascular SMCs (Figs. 1 and 2). Immunohistochemical analysis revealed that in 10-day-old embryos, a-SM actin was weakly de-

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FIG. 8. Characterization of the negative element by gel-shift assay and effect of forced expression of MSSP-1 on the a-SM actin promoter activity. Analysis of transacting factors to the negative element (A). A 20-bp DNA fragment expanding from 2238 to 2219, NE20 (Fig. 7A) was end-labeled by [g- 32P]ATP and used as a probe. In this assay, a 22-bp DNA fragment containing the CArG A (Fig. 7A) was used as a nonspecific competitor. 32P-labeled NE20 was reacted with 4 mg of nuclear extracts from differentiated (lanes 1 to 3) or dedifferentiated (lanes 4 to 6) SMCs without a competitor (lanes 1 and 4), with a 50-fold excess of unlabeled NE20 (lanes 2 and 5) or with a 50-fold excess of unlabeled nonspecific DNA (ns) (lanes 3 and 6). Nonspecific DNA-protein complexes are marked by asterisk. The effect of forced expression of MSSP-1 on the promoter activity of pActCAT238 (B). PActCAT238 or pActCAT238 (NEmut) were cotransfected with pEF-MSSP-1 or pEF control vector in dedifferentiated gizzard SMCs. The promoter activities of respective transfectants are graphically presented. Normalization of the activities was described in the legend of Fig. 6.

tected in aortic media, while this protein dramatically increased in association with aortic development. In 10-day-old embryos, the expression of a-SM actin was restricted in the smooth muscles of visceral tissues such as gizzard, trachea, and intestine, except for aortic media, and decreased thereafter. The opposite pattern of a-SM actin expression was similarly found in developing rat visceral and vascular smooth muscle

tissues (data not shown). The paradoxical expression of a-SM actin in both different smooth muscles is, therefore, considered to be a common molecular event in vertebrates. Morphological studies demonstrated that during embryogenesis, massively proliferating mesenchymal cells developed into gizzard SMCs [34, 35]. The 10-day-old embryo is critical for phenotypic modulation of gizzard cells from undifferentiated to differentiated SMCs. In developing aorta, the medial mesenchymal cells gradually change in their phenotype to differentiated SMCs [36]. Combined with these morphological observations, our present results suggest that the upand the downregulations of a-SM actin oppositely correlate with phenotypic modulation of vascular and visceral SMCs from undifferentiated to differentiated states, respectively. Skalli et al. [2] have demonstrated that the a-SM actin staining is negative in gizzard muscles in addition to tracheal and intestinal SMC layers of adult chicken. We confirmed their findings except for the inner layer of intestinal muscle layers, in which a-SM actin is intensely expressed in 15-day-old embryos and 1-month-old chickens after hatching. The muscularis mucosae of adult rat intestine has been reported to be stained with the same antibody [2]. These findings suggest the presence of two different SMC lineages in intestines. To characterize SMC phenotype-dependent expression of a-SM actin, we introduced a unique culture system of gizzard SMCs maintaining a differentiated state for a long culture (Fig. 3). Based on cell morphology, function, and expression of SMC-specific molecular markers, we concluded that SMCs cultured on laminin under insulin-stimulated conditions showed a fully differentiated phenotype. Browning et al. have recently reported a primary culture of gizzard SMCs on collagen type-IV in the presence of insulin [37]. Collagen type-IV was less potent than laminin for delaying the progression of SMC dedifferentiation under nonstimulated conditions (Fig. 3A), while insulin made it possible to maintain a differentiated phenotype of SMCs when they were cultured not only on laminin (Figs. 3B and 3C) but also on other ECM components including collagen type-IV (data not shown). Under the present culture conditions, SMCs expressed SMC-specific molecular markers at mRNA and protein levels (Figs. 3B and 4B). By contrast, dedifferentiation of visceral SMCs in primary culture showed the upregulation of a-SM actin expression (Figs. 5B and 5C). A change in the a-SM actin expression would be caused by phenotypic modulation of SMCs themselves, but not due to a replacement of differentiated SMCs by proliferated fibroblasts derived from contaminated connective tissue and serosa, because immunofluorescence microscopy revealed that stress fibers in chick embryo fibroblasts (CEFs) were not labeled with anti-a-SM actin anti-

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FIG. 9. Binding of SRF to the CArGs A and B and specific binding of a nuclear protein factor to the purine-rich motif. A 22-bp DNA fragment containing the CArG A (2126 to 2105) or the CArG B (276 to 255) (Fig. 7A) was equally end-labeled by [g- 32P]ATP and used as probes (A) and (B). A 23-bp DNA fragment containing the purine-rich motif (in Fig. 7A), Pu23, was equally end-labeled and used as a probe (C). (A) Four micrograms of nuclear extracts from dedifferentiated (lanes 1 and 3) or differentiated (lanes 2 and 4) SMCs were incubated with indicated probes. (B) Respective probe and 4 mg of nuclear extracts from differentiated SMCs were incubated with 300 mg of nonimmune rabbit antibodies (lanes 2 and 5), equal amount of anti-SRF antibodies (lanes 3 and 6), or without antibodies (lanes 1 and 4). Since the affinity of SRF to the CArG A was weak compared with that to the CArG B, the radioactivity of the CArG A was several-fold higher than that of the CArG B in supershift assays. (C) Four micrograms of nuclear protein extracts from differentiated (lanes 1 to 3) or dedifferentiated (lanes 4 to 6) SMCs was incubated with the probe without a competitor (lanes 1 and 4), with a 50-fold excess of unlabeled Pu23 (lanes 2 and 5) or with a 50-fold excess of unlabeled Pu23mut (lanes 3 and 6).

body, but those in dedifferentiated SMCs were clearly stained [12]. This conclusion would also be justified by the findings that the expression of a-SM actin mRNAs and protein began to occur earlier than cell proliferation (Figs. 5A–C). The a-SM actin promoters have been extensively studied, and cell type-specific cis-elements have been identified [29 –31, 38 – 41]. These studies using passaged vascular SMCs and myogenic or fibroblastic cell lines have revealed that purine-rich motif [30] and two

CArG box-like motifs [31] are positive elements and that TEF1 and SRF are their transacting factors, respectively. Promoter analysis in this paper is the first demonstration to compare the transcriptional regulation of a-SM actin gene in both differentiated and dedifferentiated phenotypes of visceral SMCs. Regarding negative regulation, Cogan et al. [30] and Sun et al. [42] have reported two protein factors (VACssBF1 and VACssBF2) specifically bound to ssDNA expanding from the E box to the purine-rich motif as repressors of

FIG. 10. Summary of phenotype-dependent transcriptional regulation of the chicken a-SM actin promoter in gizzard SMCs. Negative and positive cis-elements involved in the phenotypic-dependent transcription in gizzard SMCs are indicated by boxes in the alignment map of the 59-upstream region of the chicken a-SM actin gene. NE and Pu mean the negative element and the purine-rich motif, respectively. Transacting factors interacting with respective cis-elements are shown by arrows. The negative and positive elements which are functionally involved in the transcriptional regulation in differentiated and dedifferentiated SMCs are marked by closed and shaded bars, respectively.

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mouse a-SM actin promoter in BC3H1 myoblasts and AKR-2B fibroblasts. We also found that respective sense and antisense strands of the Pu23 containing the purine-rich motif formed multiple sequence-specific ssDNA-protein complexes (data not shown). These transacting factors may be VACssBF1 and VACssBF2. As demonstrated in this paper, these ssDNA-binding factors were, however, not directly involved in the negative regulation of a-SM actin promoter in gizzard SMCs, because pActCAT193 containing the binding sites of these factors showed the highest promoter activity (Fig. 6). In this study, we identified a distinct sequence expanding from 2238 to 2219 as a novel negative element and found protein factors in SMC nuclear extracts which specifically interacted with this element. The protein factors in dedifferentiated SMC nuclear extracts formed a more intense complex with the negative element than those from differentiated SMC nuclear extracts (Fig. 8A). In our recent study, we have identified one of these factors as MSSP-1 which binds to both sense and antisense strands of NE20 [32]. Forced expression of MSSP-1 in dedifferentiated SMCs induced the reduction of a-SM actin promoter activity, suggesting that this factor functionally acts as a suppressor of a-SM actin promoter in a sequence-specific manner (Fig. 8B). Further study regarding the molecular mechanism of transcriptional suppression mediated by MSSP-1 is required. We presently speculate that the binding of MSSP-1 to the negative element may stabilize a partial single-stranded DNA structure in the a-SM actin promoter and inhibit the interaction of positive regulators, TEF-1 and SRF, with the downstream positive elements, purine-rich motif and CArG boxes. In regard to the cell type-specific expression of a-SM actin, MSSP-1 may inhibit the transcription in other nonmuscle cells because MSSP-1 is ubiquitously expressed. In dedifferentiated SMCs, the positive regulatory factors, TEF-1 and SRF, might overcome the negative effect of MSSP-1 because significant difference in the expression of MSSP-1 was not observed in both differentiated and dedifferentiated SMCs (data not shown) and the nuclear extracts from dedifferentiated SMCs formed a more intense complex than those from differentiated SMCs (Fig. 8A). The activation of the promoter in dedifferentiated SMCs strongly depends on the purine-rich motif plus the CArGs A and B, whereas the CArG B solely functions as a positive element in differentiated SMCs (Fig. 7). The interaction of SRF with the CArGs A and B was extremely weak in nuclear extracts from dedifferentiated SMCs (Fig. 9A), whereas there was no drastic change in the expression levels of SRF in differentiated and dedifferentiated SMCs [13, 25]. In spite of such difference, dedifferentiated SMCs showed high levels of a-SM actin promoter activity, but differentiated

SMCs did not. This discrepancy suggest that additional factor(s) in association with SRF would be involved in SMC phenotype-dependent transcription of this gene. Owens’s group has recently reported that homeodomian factor, MHox, enhances the binding of SRF to the CArGs A and B in the rat a-SM actin promoter and transactivates a a-SM actin promoter/CAT construct in cultured aortic SMCs [43]. Considering these findings, MHox or other SMC-specific homeodomain factor(s) might be involved in SMC phenotype-dependent transcription of the a-SM actin gene. Based on our present results, we summarized the transcriptional regulation of the a-SM actin gene in different phenotypes of visceral SMCs (Fig. 10). The overall promoter activity in differentiated SMCs would be dominantly suppressed by the negative element even though the CArG B functionally achieves its role. On the other hand, in dedifferentiated SMC, the enhanced promoter activity by the three positive elements would overcome the suppression by the negative element. The promoter is, therefore, totally active. Thus, changes in the balance of the positive and negative cis-elements and their respective transacting factors might determine the opposite expression of a-SM actin in visceral and vascular SMCs in their phenotype-dependent manner. The regulation of a-SM actin expression is a useful parameter for the molecular approach of pathogenesis such as atherosclerosis and glomerulonephritis involving massive proliferation of mesangial cells. In the latter case, the expression of a-SM actin is strongly upregulated in mesangial cells [10] and this upregulation is due to activation of a-SM actin promoters [44]. Therefore, the transcriptional regulation of the a-SM actin gene is also important for revealing the pathogenetic mechanism of such impairment. We thank Dr. H. Ariga (Faculty of Pharmaceutical Sciences, Hokkaido University) for providing an MSSP-1 expression plasmid. This work was supported to K.S. by Grants-in-Aid for COE research from the Ministry of Education, Science, Sports, and Culture of Japan.

REFERENCES 1.

Vandekerckhove, J., and Weber, K. (1978). At least six different actins are expressed in a higher mammal: An analysis based on the amino acid sequence of the amino-terminal tryptic peptide. J. Mol. Biol. 126, 783– 802. 2. Skalli, O., Ropraz, P., Trzeciak, A., Benzonana, G., Gillessesn, D., and Gabbiani, G. (1986). A monoclonal antibody against a-smooth muscle actin: A new probe for smooth muscle differentiation. J. Cell Biol. 103, 2787–2796. 3. Owens, G. K., and Thompson, M. M. (1986). Developmental changes in isoactin expression in rat aortic smooth muscle cells in vivo. J. Biol. Chem. 261, 13373–13380. 4. Glukhova, M., Frid, M. G., and Koteliansky, V. E. (1990). Developmental changes in expression of contractile and cytoskeletal proteins in human aortic smooth muscle. J. Biol. Chem. 265, 13042–13046.

SMOOTH MUSCLE ACTIN 5.

Campbell, J. H., Kocher, O., Skalli, O., Gabiani, G., and Campbell, G. R. (1989). Cytodifferentiation and expression of a-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells. Arteriosclerosis 9, 633– 643.

6.

Blank, S. R., Thompson, M. M., and Owens, G. K. (1988). Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells. J. Cell Biol. 107, 299 –306.

7.

Izant, J. G., and Lazarides, E. (1977). Invariance and heterogeneity in the major structural and regulatory proteins of chick muscle cells revealed by two-dimensional gel electrophoresis. Proc. Natl. Acad. Sci. USA 74, 1450 –1454.

8.

Saborio, J. L., Segura, M., Flores, M., Garcia, R., and Palmer, E. (1979). Differential expression of gizzard actin genes during chick embryogenesis. J. Biol. Chem. 254, 11119 –11125.

9.

Lazard, D., Sastre, X., Frid, M. G., Glukhova, M. A., Thiery, J. P., and Koteliansky, V. E. (1993). Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue. Proc. Natl. Acad. Sci. USA 90, 999 –1003.

10.

11.

12.

13.

14.

15.

16.

17.

18.

Johnson, R. J., Iida, H., Alpers, C. E., Majesky, M. W., Schwartz, S. M., Pritzl, P., Gordon, K., and Gown, A. M. (1991). Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. J. Clin. Invest. 87, 847– 858. Kirk, T. Z., Mark, M. E., Chua, C. C., Chua, B. H., and Mayes, M. D. (1995). Myofibroblasts from scleroderma skin synthesize elevated levels of collagen and tissue inhibitor of metalloproteinase (TIMP-1) with two forms of TIMP-1. J. Biol. Chem. 270, 3423–3428. Kashiwada, K., Nishida, W., Hayashi, K., Ozawa, K., Yamanaka, Y., Saga, H., Yamashita, T., Tohyama, M., Shimada, S., Sato, K., and Sobue, K. (1997). Coordinate expression of a-tropomyosin and caldesmon isoforms in association with phenotypic modulation of smooth muscle cells. J. Biol. Chem. 272, 15396 –15404. Obata, H., Hayashi, K., Nishida, W., Momiyama, T., Uchida, A., Ochi, T., and Sobue, K. (1997). Smooth muscle cell phenotypedependent transcriptional regulation of the a1 integrin gene. J. Biol. Chem. 270, 26643–26651. Hirai, S., and Hirabayashi, T. (1983). Developmental change of protein constituents in chicken gizzards. Dev. Biol. 97, 483– 493. Hayashi, K., Yamada, S., Kanda, K., Kimizuka, F., Kato, I., and Sobue, K. (1989). Primary structure and functional expression of h-caldesmon complementary DNA. Biochem. Biophys. Res. Commun. 164, 503–511.

19.

20.

21.

22.

23.

24.

25.

26.

27. 28.

29.

30.

31.

Sobue, K., Kanda, K., Miyamoto, I., Iida, K., Yahara, I., Hirai, R., and Hiragun, A. (1989). Comparison of the regional distribution of calspectin (nonerythroid spectrin or fodrin), a-actinin, vinculin nonerythroid protein 4.1, and calpactin in normal and avian sarcoma virus- or rous sarcoma virus-induced transformed cells. Exp. Cell Res. 181, 256 –262.

32.

Takahashi, K., Hiwada, K., and Kokubo, T. (1987). Occurrence of anti-gizzard p34K antibody cross-reactive components in bovine smooth muscles and non-smooth muscle tissues. Life Sci. 41, 291–296.

33.

Carroll, S. L., Bergsma, D. J., and Schwartz, R. J. (1988). A 29-nucleotide DNA segment containing and evolutionarily conserved motif is required in cis for cell-type-restricted repression of chicken a-smooth muscle actin gene core promoter. Mol. Cell. Biol. 8, 241–250.

34.

291

Takenaka, M., Noguchi, T., Inoue, H., Yamada, K., Matsuda, T., and Tanaka, T. (1989). Rat pyruvate kinase M gene. J. Biol. Chem. 264, 2363–2367. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2, 1044 –1051. Yano, H., Hayashi, K., Momiyama, T., Saga, H., Haruna, M., and Sobue, K. (1995). Transcriptional regulation of the chicken caldesmon gene. J. Biol. Chem. 270, 23661–23666. Negishi, Y., Nishita, Y., Saegusa, Y., Kakizaki, I., Galli, I., Kihara, F., Tamai, K., Miyajima, N., Iguchi Ariga, S. M., and Ariga, H. (1994). Identification and cDNA cloning of single-stranded DNA binding proteins that interact with the region upstream of the human c-myc gene. Oncogene 9, 1133–1143. Iida, M., Taira, T., Ariga, H., and Iguchi Ariga, S. M. (1997). Induction of apoptosis in HeLa cells by MSSP, c-myc binding proteins. Biol. Pharm. Bull. 20, 10 –14. de Wet, J. R., Wood, K. V., Deluca, M., Helinski, D. R., and Subramani, S. (1987). Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725–737. Momiyama, T., Hayashi, K., Obata, H., Chimori, Y., Nishida, T., Ito, T., Kamiike, W., Matsuda, H., and Sobue, K. (1998). Functional involvement of serum response factor in the transcriptional regulation of caldesmon gene. Biochem. Biophys. Res. Commun. 242, 429 – 435. Chaponnier, C., Goethals, M., Janmey, P. A., Gabbiani, G., and Vandekerckhove, J. (1995). The specific NH2-terminal sequence Ac-EEED of a-smooth muscle actin plays a role in polymerization in vitro and in vivo. J. Cell Biol. 130, 887– 895. Chamley-Campbell, J., Campbell, G. R., and Ross, R. (1979). The smooth muscle cell in culture. Physiol. Rev. 59, 1– 6. Hedin, U., Bottger, B. A., Forsberg, E., Johansson, S., and Thyberg, J. (1988). Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J. Cell. Biol. 107, 307–319. Carroll, S. L., Bergsma, D. J., and Schwartz, R. J. (1986). Structure and complete nucleotide sequence of the chicken a-smooth muscle (aortic) actin gene. J. Biol. Chem. 261, 8965– 8976. Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995). Plasticity of vascular smooth muscle a-actin gene transcription. J. Biol. Chem. 270, 11310 –11321. Shimizu, R. T., Blank, R. S., Jervis, R., Lawrenz-Smith, S. C., and Owens, G. K. (1995). The smooth muscle a-actin gene promoter is differentially regulated in smooth muscle versus nonsmooth muscle cells. J. Biol. Chem. 270, 7631–7643. Kimura, K., Saga, H., Hayashi, K., Obata, H., Chimori, Y., Ariga, H., and Sobue, K. (1998). c-Myc gene single-strand binding protein-1, MSSP-1, suppresses transcription of a-smooth muscle actin gene in chicken visceral smooth muscle cells. Nucleic Acids Res. 26, 2420 –2425. Sawtell, N. M., and Lessard, J. L. (1989). Cellular distribution of smooth muscle actins during mammalian embryogenesis: Expression of the a-vascular but not the enteric isoform in differentiating striated myocytes. J. Cell Biol. 109, 2929 – 2937. Bennett, T., and Cobb, J. L. S. (1969). Studies on the avian gizzard: The development of the gizzard and its innervation. Z. Zellforsch. 98, 599 – 621.

292 35.

36.

37.

38.

39.

40.

SAGA ET AL. Chou, R. G. R., Stromer, M. H., Robson, R. M., and Huiatt, T. W. (1992). Assembly of contractile and cytoskeletal elements in developing smooth muscle cells. Dev. Biol. 149, 339 –348. Kocher, O., Skalli, O., Cerutti, D., Gabbiani, F., and Gabbiani, G. (1985). Cytoskeletal features of rat aortic cells during development. Circ. Res. 56, 829 – 838. Browning, C. L., Culberson, D. E., Aragon, I. V., Fillmore, R. A., Croissant, J. D., Schwartz, R. J., and Zimmer, W. E. (1998). The developmentally regulated expression of serum response factor plays a key role in the control of smooth muscle-specific genes. Dev. Biol. 194, 18 –37. Min, B., Foster, D. N., and Strauch, A. R. (1990). The 59flanking region of the mouse vascular smooth muscle a-actin gene contains evolutionarily conserved sequence motifs within a functional promoter. J. Biol. Chem. 265, 16667–16675. Nakano, Y., Nishihara, Y., Sasayama, S., Miwa, T., Kamada, S., and Kakunaga, T. (1991). Transcriptional regulatory elements in the 59 upstream and first intron regions of the human smooth muscle (aortic type) a-actin-encoding gene. Gene 99, 285–289. Blank, S. R., McQuinn, T. M., Yin, K. C., Thompson, M. M., Takeyasu, K., Schwartz, R. J., and Owens, G. K. (1992). Ele-

Received March 2, 1998 Revised version received August 31, 1998

41.

42.

43.

44.

ments of the smooth muscle a-actin promoter required in cis for transcriptional activation in smooth muscle. J. Biol. Chem. 267, 984 –989. Foster, D. N., Min, B., Foster, L. K., Stoflet, E. S., Sun, S., Getz, M. J., and Strauch, A. R. (1992). Positive and negative cisacting regulatory elements mediate expression of the mouse vascular smooth muscle a-actin gene. J. Biol. Chem. 267, 11995–12003. Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995). Negative regulation of the vascular smooth muscle a-actin gene in fibroblasts and myoblasts: Disruption of enhancer function by sequence-specific single-stranded-DNAbinding proteins. Mol. Cell. Biol. 15, 2429 –2436. Hautmann, M. B., Thompson, M. M., Swartz, E. A., Olson, E. N., and Owens, G. K. (1997). Angiotensin II-induced stimulation of smooth muscle a-actin expression by serum response factor and homeodomain transcription factor MHox. Circ. Res. 81, 600 – 610. Simonson, M. S., Walsh, K., Kumar, C. C., Bushel, P., and Herman, W. (1995). Two proximal CArG elements regulate SM a-actin promoter, a genetic marker of activated phenotype of mesangial cells. Am. J. Physiol. 268, F760 –F769.