Differential activation of the SMalpha A promoter in smooth vs. skeletal muscle cells by bHLH factors

A. Daniel Johnson1 and Gary K. Owens2

1 Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109; and 2 Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

E-box/basic helix-loop-helix (bHLH)-dependent regulation of promoters for skeletal muscle-specific genes is well established, but similar regulation of smooth muscle-selective promoters has not been reported. Using transient transfection assays of smooth muscle alpha -actin (SMalpha A) promoter-chloramphenicol acetyltransferase (CAT) reporter constructs in rat vascular smooth muscle cells (SMCs) and L6 skeletal myotubes, we identified two activator elements, smE1 and smE2, with sequences corresponding to E-box (5'-CAnnTG-3') motifs. In L6 myotubes, 4-bp mutations of smE1 or smE2 E-box motif alone completely abolished promoter activity. In contrast, mutation of smE1 and smE2 was required to reduce promoter activity in SMCs. Supershift analyses identified a myogenin-containing complex as the predominant smE1 and smE2 binding activity in skeletal muscle, and myogenin overexpression transactivated the promoter. Supershift analyses with SMC extracts demonstrated that the bHLH protein upstream stimulatory factor (USF) bound smE1, and USF overexpression transactivated the promoter in an smE1-dependent manner. In summary, our results provide novel evidence implicating E-box elements in directing expression of the SMalpha A promoter through distinct bHLH factor complexes in skeletal vs. smooth muscle.

alpha -actin promoter; basic helix-loop-helix protein; E-box; upstream stimulatory factor; myogenin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

SMOOTH MUSCLE alpha -actin (SMalpha A) is the earliest known marker of differentiated smooth muscle cells (SMCs) expressed during development of the arterial wall (21). Conversely, reduced expression of SMalpha A is a hallmark of the vascular response to injury and of the relatively dedifferentiated SMCs present in atherosclerotic lesions (34, 35). Activity of the SMalpha A promoter appears to be closely (but not inseparably) linked to expression of the larger repertoire of proteins that are characteristic of the differentiated phenotype of SMCs.

The precise mechanisms for transcriptional regulation of the SMalpha A promoter in vascular SMCs remain unknown. A number of cis elements have been identified in the SMalpha A promoter that contribute to tissue-specific expression patterns. These include two separate CArG motifs (7) and a transforming growth factor-beta (TGF-beta ) controller element (17). However, much remains to be elucidated about how these and other unidentified elements within the promoter regulate specificity of expression in SMCs as well as alterations in their response to injury or atherogenic stimuli.

Expression of the SMalpha A gene is not restricted to SMCs alone. Although expressed primarily in differentiated SMCs of adult animals, SMalpha A is also transiently expressed in fibroblasts, cardiomyocytes, and skeletal muscle cells during development, after tissue wounding, or in culture (4, 10, 25, 37, 41, 42). Expression of SMalpha A by all three muscle types suggests that the SMalpha A promoter may be activated by a similar set of transcription factors and cis elements, such as binding of basic helix-loop-helix (bHLH) transcription factors to E-box elements. The E-box motif (5'-CAnnTG-3') binds dimers of bHLH transcription factors (30). Tissue-specific bHLHs are exemplified by the MyoD family (including Myf-5, Mrf-4, and myogenin) and are the primary transcription factors coordinating expression of skeletal muscle-restricted genes (13). Similarly, cardiac development is partially regulated by the bHLH factors Th-1/dHAND and Th-2/eHAND (9, 18, 45). Similar to striated muscle, several widely expressed bHLH factors, including E12/E47 (22, 36), Id (24, 28), and members of the c-Myc-related, bHLH-leucine zipper (bHLH-ZIP) family (3, 12, 44) are expressed in SMCs. General mesodermal and neuroectodermal lineage-determinant bHLH factors such as M-twist, Meso-1, and HES-1-5 (6, 23, 39, 48) also appear transiently in the aortic primordia. In contrast to striated muscle, however, regulation by bHLH factors of a gene associated with the SMC-differentiated phenotype has not been demonstrated (35). Deletion analyses of the rat SMalpha A promoter by our laboratory showed that the first 125 bp (-1 to -125) 5' to the transcriptional start site drove high-level expression of a linked chloramphenicol acetyltransferase (CAT) reporter gene in transient transfection studies with cultured aortic SMCs (28, 41) but not with L6 skeletal myoblasts or myotubes. Additional promoter sequences from -125 to -271 bp resulted in substantial reporter expression in L6 myotubes but decreased activity by one-half in SMCs. Thus positive and negative regulatory elements are present in the rat SMalpha A promoter between -125 and -271 bp. Several candidate regulatory elements reside within this region, including three potential elements at -214 bp (smE1), -252 bp (smE2), and -260 bp (smE3) that correspond to the canonical E-box motif. In the SMalpha A promoters of other species, smE1 and smE2 are highly conserved, whereas the putative E-box at smE3 is present in the rat promoter only (7, 41).

Given the general importance of E-box elements in differentiation of striated muscle and their presence in a transcriptionally active region of the SMalpha A promoter, the goals of this study were 1) to determine whether the three 5'-CAnnTG-3' motifs within the SMalpha A promoter function as true E-boxes within the context of rat L6 skeletal myotubes and/or rat aortic SMCs and, if so, 2) to identify transcription factors that bind the active sites. Our results demonstrated that, in skeletal myotubes, smE1 and smE2 elements were required for SMalpha A promoter activity and bound myogenin-containing transcription factor complexes present in L6 myotube nuclear extracts. Furthermore, myogenin overexpression could transactivate the SMalpha A promoter. In SMCs, smE1 and smE2 could mutually compensate for each other in transient transfection assays but bound different transcription factor complexes. smE1 acted as an E-box that was recognized by two bHLH factors, upstream stimulatory factors USF-1 and USF-2, and overexpression of USFs could transactivate the SMalpha A promoter in an smE1-dependent manner. In contrast, mutagenesis data indicated that smE2 did not appear to function as an E-box in SMCs, whereas data from gel-shift analyses indicated that smE2 was not bound by any of several bHLH factors, including USFs. Our data are the first to demonstrate regulation of an SMC differentiation marker gene via an E-box-dependent mechanism.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell culture. Rat aortic SMCs and endothelial cells were cultured as described previously (41). All other cell lines were obtained from American Type Culture Collection (Manassas, VA). L6 myoblasts were routinely maintained in Ham's F-12 + 10% FCS and fused into myotubes as needed using DMEM-1% FCS for 48 h. COS-7 cells were grown in DMEM + 10% FCS. Rat PC-12 pheochromocytoma cells were maintained in RPMI 1640 + 10% horse serum and 5% FCS. 10T1/2 cells were routinely grown in basal medium Eagle-10% FCS.

Gel-shift analysis of E-box binding proteins. Nuclear and whole cell extracts were prepared as described previously (11, 29). Double-stranded oligonucleotides containing 6 bp of native or mutant smE1, smE2, or smE3 plus 5 bp of native SMalpha A promoter sequence 5' and 3' to each E-box were end labeled with 32P and purified. DNA-protein binding reactions were separately optimized for L6 skeletal myotube and SMC nuclear extracts. SMC binding reactions contained 5 µg of nuclear extract protein and 2 × 105 cpm of DNA probe in a 20-µl final reaction volume of 15 mmol/l HEPES, pH 7.9, 80 mmol/l NaCl, 25 mmol/l KCl, 1 mmol/l EDTA, 1.25 mmol/l CaCl2, 1.2 mmol/l dithiothreitol, 15% glycerol, 200 µg/ml BSA, 0.05 mmol/l 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.2 µg/ml each aprotinin and leupeptin, and 0.2 µg of polydeoxyinosinic-deoxycytidylic acid. For L6 myotubes, binding was optimal in 10 mmol/l Tris, pH 7.5, 100 mmol/l KCl, 1 mmol/l EDTA, 1 mmol/l dithiothreitol, and 5% glycerol, with 2 µg of polydeoxyinosinic-deoxycytidylic acid. In cold competitor assays, 10- to 1,000-fold molar excess unlabeled oligonucleotide was added to the binding reaction 30 min before the radiolabeled probe. Samples were then resolved on 5% 29:1 acrylamide-bis-0.5× Tris base-EDTA-boric acid gels. For supershift assays, 1-2 µg of antibodies against candidate bHLH proteins were incubated with standard gel-shift reactions for 30 min before loading. Antibodies were as follows: Yae monoclonal antibody against E2A gene products, A20 polyclonal antibody against HEB, C33 monoclonal and C17 polyclonal antibodies against c-Myc and Max, respectively, M-225 polyclonal antibody against myogenin, and C20 or N18 polyclonal antibodies against USF-1 and USF-2 (all from Santa Cruz Biotechnologies, Santa Cruz, CA), G108-391 monoclonal antibody against human E2-2 (Pharmingen, San Diego, CA), and an additional antibody against murine E2-2-related gene products (1).

Site-directed mutagenesis of -271-bp SMalpha A promoter-CAT reporter and functional analysis by transient transfection. To determine whether the putative E-boxes at -214, -252, and -260 bp of the rat SMalpha A promoter are required for transcriptional activity, 1- to 4-bp mutations were made within each of the three sites by use of the Altered Sites protocol (Promega, Madison, WI) and PCR (19). The -271- to +43-bp region of native or E-box mutant promoters was then subcloned into the Hind III/Sal I sites of pCAT.Basic reporter (Promega; Fig. 1). SMCs and L6 myotubes were then transfected with CsCl-purified DNA constructs, and CAT reporter assays were performed as described previously (41). Briefly, cells were plated at 2 × 104 cells/cm2 in six-well plates; SMCs were plated 24 h before transfection in DMEM-10% FCS, and undifferentiated myoblasts were plated 72 h before transfection in Ham's F-12-10% FCS. At time 0, cells were washed three times with serum-free DMEM, then transfected with 4 µg of DNA construct mixed with 30 µg of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (Boehringer Mannheim, Indianapolis, IN) in 50 mM HEPES buffer, pH 7.9. In all assays the promoterless pCAT-Basic plasmid and the simian virus 40 (SV40)-driven constitutively active pCAT.Control plasmid were included as baseline and positive controls for the transfection and CAT assays. Five hours after addition of DNA, cells were refed serum-containing medium to a final concentration of 10% FCS for SMCs or 1% for L6 myoblasts. For L6 myoblasts, this reduction in serum concentration was sufficient to induce differentiation into myotubes. After 18 h, SMCs or L6 myotubes were refed fresh DMEM plus 10% or 1% FCS, respectively. Cells were harvested, postnuclear lysates were prepared 72 h after transfection, and total protein in lysates was determined by the Bradford method (Bio-Rad, Hercules, CA). CAT expression per milligram of protein in the postnuclear cell lysate was determined as described previously (41), and reporter activity was normalized to a percentage of pCAT.Basic activity. Three replicates per DNA construct were assayed in each experiment, with each experiment replicated at least twice. In all cases, CAT activity for test reporter constructs was less than the expression observed for cells transfected with the SV40-driven CAT-positive control.




View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1.   A: schematic diagram of pCAT.271/native construct, consisting of -271 to +43 bp of smooth muscle alpha -actin (SMalpha A) promoter cloned into pCAT.Basic. CAT, chloramphenicol acetyltransferase; SV40, simian virus 40. B and C: effects of E-box-disrupting mutations on expression levels of pCAT.271 in L6 myotubes and smooth muscle cells (SMCs), respectively. Cells were transfected with 4 µg of promoter-reporter construct containing 4-bp mutations listed in Table 1. At 48 h after transfection, cells were lysed and CAT reporter activity was determined. Expression levels are means ± SD of 3 independent experiments, with triplicate samples per experimental group, normalized to pCAT.Basic = 100%. * Significantly different (P < 0.05) from pCAT.271/native promoter.

Interestingly, SMCs exhibited some time-dependent differences in activity of the native promoter construct (Fig. 1C vs. Fig. 2). We have found this a puzzling occurrence, since it reflects data obtained using different established primary SMC lines. Importantly, the quantitative differences between experimental groups were identical between multiple replicate experiments performed with a single primary cell line. Moreover, the relative differences between experimental groups was reproducible between multiple independently derived lines. Thus we are confident of the reproducibility of the findings.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Mutagenesis analysis of nucleotides in smE1 vs. smE2 required for SMalpha A promoter activity in SMCs. Cells were transfected with native or E-box mutant pCAT.271 constructs, and CAT reporter activity was assayed as described in Fig. 1 legend. Left: specific mutations of smE1 or smE2 sequences. Nucleotides differing from native promoter sequences are shown, with retained native sequence indicated by dashes. Middle: smE1 mutations; bottom: smE2 mutations. Promoter activity is normalized to pCAT.Basic = 100%. Values are means ± SD of 3 independent experiments done in triplicate. * Significantly different (P < 0.05) from pCAT.271/native promoter.

Transactivation of SMalpha A promoter by USF or myogenin overexpression. To determine whether overexpression of USFs or myogenin increased SMalpha A promoter activity, SMCs or 10T1/2 fibroblasts were cotransfected with native or E-box-mutated CAT constructs plus USF-1, USF-2, or myogenin expression plasmids. The 1.7-kb EcoR I fragment encoding human USF-1 from plasmid d12 and the 2-kb fragment encoding murine USF-2 from pM2-2 (26, 27) were subcloned into pcDNA3.1+ (InVitrogen, San Diego, CA) under control of the cytomegalovirus (CMV) promoter or pSG-5 (Stratagene, La Jolla, CA) under the SV40 promoter. Myogenin in pEMSV is an SV40-driven constitutive expression vector. For SMC cotransfection assays, 1 µg of either empty vector (baseline control), USF-1, or USF-2 expression constructs was mixed with 4 µg of pCAT.271/native, smE1 mutant, or smE2 mutant promoter constructs, and cells were transfected using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate. For transactivation assays in 10T1/2 cells, 1.9 × 105 cells in six-well plates were transfected with 3 µg of reporter constructs plus 1 µg of myogenin or empty expression vectors with use of calcium phosphate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutations of putative E-boxes in the SMalpha A promoter differentially inhibited transcriptional activity in L6 myotubes vs. SMCs. A schematic of the pCAT.271/native construct containing 3 putative SMalpha A promoter E-box elements, designated smE1, smE2, and smE3, is shown in Fig. 1A; native vs. mutated sequences are listed in Table 1. Four-base pair mutations were generated in each of the three E-boxes, which, on the basis of previous studies (5, 13, 20, 23, 30, 46), would maximally disrupt E-box interactions with bHLH factors. Effects of these mutations on promoter activity were assayed by transient transfection into rat aortic SMCs and L6 skeletal myotubes. As shown in Fig. 1B, disruption of smE1 or smE2 alone nearly abolished CAT reporter activity in L6 myotubes, whereas mutation of smE3 had no consistent effect. In contrast to skeletal myotubes, disruption of smE1 or smE2 alone had no significant effect on CAT activity in SMCs (Fig. 1C). Mutation of smE3 alone did not produce consistent effects on CAT activity; the 4-bp smE3 mutation modestly increased promoter activity (as shown) or, equally often, had no effect on activity (data not shown).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   SMalpha A promoter mutants tested in CAT reporter and gel shift assays

Although the functional importance of bHLH factors binding to paired E-boxes is well documented for promoter activation in L6 myotubes and other skeletal muscle contexts (1, 13, 33), similar bHLH-dependent promoter regulation has not been previously established in an SMC context. Indeed, our initial results indicated that functional disruption of the E-boxes corresponding to smE1 or smE2 alone had no effect on SMalpha A promoter activity. However, previous studies (32, 38) have also shown that although the skeletal and cardiac alpha -actin promoters contain paired E-boxes, transcriptional activity can be maintained by a single E-box interacting with serum response factor or Sp1 binding sites. Therefore, we also tested the effects of combining the 4-bp mutations that caused functional disruption of smE1 or smE2 with additional 1- to 2-bp mutations that disrupted the 5'-conserved, 3'-conserved, or internal dinucleotide regions of the second potential E-box element.

As shown in Fig. 2, promoter activity was relatively unaffected by mutation of the internal dinucleotide region of smE1 (i.e., GC to TA) in the context of functional disruption of smE2. In contrast, mutation of the 3'-conserved TG sequence (to AT) reduced promoter activity by ~30%, whereas mutation of the 5'-conserved CA sequence (to AC) reduced promoter activity by 40% in SMCs. Notably, however, neither the 5'-CA nor the 3'-TG mutation completely abolished promoter activity. This suggests that the smE1 region is not functioning as a strict, canonical E-box in SMCs or that bHLH proteins interacting with the smE1 region exhibit relaxed binding site sequence requirements. In contrast to smE1, a GT-to-GA mutation of the central dinucleotide of smE2 significantly reduced promoter activity in SMCs, whereas mutations of the 5'-CA- or 3'-TG-conserved bases were without effect. Thus conservation of a canonical E-box motif at smE2 was not required for promoter activity in SMCs. This suggests that the activity of smE2 in SMCs is regulated by interaction with bHLH factors that exhibit extremely relaxed binding specificity or, more likely, by a non-bHLH factor. Finally, paired mutations of smE1 or smE2 plus the nonconserved smE3 region showed no effect whatsoever on promoter activity (data not shown), suggesting that smE3 is not a functional cis element.

Identification of distinct smE1 and smE2 binding protein complexes in skeletal vs. smooth muscle cells by gel-shift analysis. To identify SMC and L6 myotube nuclear proteins that bound to smE1 and smE2, gel-shift assays were performed with double-stranded oligonucleotides containing native or mutated smE1 or smE2 sequences alone. L6 myotube nuclear extracts formed DNA-protein complexes with smE1- or smE2-containing oligonucleotides that exhibited identical electrophoretic mobilities, and smE1 and smE2 binding proteins were competed by an excess of unlabeled probe (Fig. 3A). Unlike smE1 and smE2, an smE3 binding complex could not be detected in nuclear extracts of skeletal myotubes. Addition of 1-2 µg of a neutralizing antibody against myogenin (one of the skeletal muscle-specific bHLH factors) inhibited formation of the major smE1 and smE2 binding complexes (Fig. 3B, smE1 data only shown), implicating myogenin as the smE1 and smE2 binding factor in L6 myotube nuclear extracts. When gel shifts were performed using the conditions described for L6 myotubes, but using SMC nuclear extracts, there was no evidence of binding by MyoD or other specific bHLH factors (data not shown).




View larger version (224K):
[in this window]
[in a new window]
 
Fig. 3.   Identification of specific smE1 and smE2 binding complexes by gel-shift analysis. SMC or L6 myotube nuclear extract proteins were mixed with radiolabeled, double-stranded oligonucleotides containing smE1 or smE2 elements plus 5 bp of surrounding sequence from the native SMalpha A promoter. Complexes were resolved on 5% acrylamide-0.5× Tris base-EDTA-boric acid gels. Cold competitions for specific binding by labeled smE1 or smE2 native oligonucleotides were performed with a molar excess of equivalent, nonradiolabeled double-stranded oligonucleotides or probes containing E-box-disrupting mutations (1M or 2M; see Table 1 for sequences). A: smE1-, smE2-, and smE3-containing oligonucleotides plus L6 myotube nuclear extracts. smE1 and smE2 each specifically bound skeletal muscle nuclear extract proteins that could be competed away by as little as 25-fold excess of unlabeled probe. Unlike smE1 and smE2 probes, smE3 probe failed to exhibit specific binding to any nuclear proteins under any conditions tested. B: 1 or 2 µg of anti-myogenin polyclonal antibody reduced smE1 complex formation, implicating myogenin as primary smE1-binding protein in these extracts under these binding conditions. In contrast, 2 µg of a nonspecific preimmune IgG (PI) did not reduce smE1 probe binding to L6 nuclear extract proteins. Similar results were obtained using smE2 probe and L6 myotube extract (data not shown). C: binding of smE1-, smE2-, or smE3-containing oligonucleotides to SMC nuclear extract proteins. Binding and competition were performed as described above. smE1 specifically bound a single protein complex in SMCs, which could be competed by 100-fold molar excess of unlabeled, native smE1 probe, but not with mutant E-box probe (1M) or a random double-stranded oligonucleotide (BS). Radiolabeled probe with a 4-bp smE1 mutation also did not bind significantly to SMC nuclear proteins (1M). smE2-specific binding was also observed, but smE2 binding complex exhibited greater electrophoretic mobility than smE1 complex. smE2 binding complex also required 1,000-fold excess specific competitor to inhibit formation. smE3 probe failed to form any specific binding complexes with SMC nuclear extract proteins; a longer overexposure of lane containing smE3 probe from same autoradiogram is shown.

A single DNA-protein complex was also observed with use of SMC nuclear extracts and the native smE1 probe. This complex was competed away with a 100-fold excess of nonradiolabeled probe (Fig. 3C), but not with a random DNA fragment (BS) or with an smE1 probe containing the same 4-bp E-box mutation tested in the CAT reporter assays. In contrast, gel-shift analyses with SMC nuclear extract and smE2-containing oligonucleotide exhibited two shift bands (Fig. 3, A and B) that were distinct from the smE1 binding complex. smE2 shift complexes never comigrated with the smE1 binding complex and could only be competed with a large (500- to 1,000-fold) excess of unlabeled double-stranded smE2 probe. Moreover, and despite extensive efforts to optimize gel-shift conditions, these bands remained somewhat diffuse in nature compared with the smE1 binding complex. The different electrophoretic characteristics and competition profiles of shift bands formed with smE1 vs. smE2 probes and SMC nuclear extracts suggested that they bound distinct protein complexes and were consistent with the differences in functional activities seen in mutational studies (Fig. 2). Unlike smE1- and smE2-containing probes, an smE3-containing probe did not form a shift band with SMC nuclear extract proteins (Fig. 3C).

To determine whether smE1 and smE2 binding factor complexes were unique to SMCs, gel shifts were performed using whole cell or nuclear extracts from multiple cell lines under gel-shift conditions previously optimized for binding of SMC factors to smE1 or smE2. As shown in Fig. 4A, all cell lines tested, including L6 myotubes, contained at least some smE1 binding activity that formed shift complexes with electrophoretic mobility similar to that observed with SMC nuclear extracts. In contrast, the mobility of smE2-binding complexes derived from SMCs (Fig. 4B, complexes A and B) differed significantly from smE2 binding proteins in the other cell lines tested. These results suggested that, unlike smE1 binding factor(s), the two smE2 binding complexes from SMCs were not present at significant levels in the other cell lines tested.



View larger version (109K):
[in this window]
[in a new window]
 
Fig. 4.   Comparison of gel-shift complexes formed with smE1 and smE2 probes in multiple cell types. Gel-shift reactions consisted of native smE1 or smE2 probes plus 5 µg of nuclear (SMC, L6, 3T3, HeLa, endothelial cell) or 10 µg of total cell (COS-7, PC-12) protein extract. Electrophoresis was done as described in Fig. 3 legend, with buffer conditions that were optimized for smE1 binding complex from SMC nuclear extracts (see EXPERIMENTAL PROCEDURES). Cell lines are indicated above each lane. A: smE1 oligonucleotide probe. smE1 binding complexes with electrophoretic mobility similar to complexes from SMCs (arrow) were present in all cell lines tested, suggesting that smE1 binding proteins are widely expressed and do not represent smooth muscle-specific factors. B: smE2 oligonucleotide probe. Multiple smE2 binding proteins were observed in SMCs and non-SMC lines, but none exhibited mobility identical to smE2 binding complex doublet (A and B) derived from SMCs.

Unlike myogenin and other bHLH factors that control skeletal muscle differentiation, there are no bHLH factors that are known to regulate vascular smooth muscle differentiation that would be candidates for smE1 or smE2 binding factors. However, even degenerate E-box elements almost invariably are bound by homo- or heterodimeric complexes containing one or more of the widely expressed type I bHLH factors. Therefore, we screened the smE1 and smE2 binding complexes for bHLH factors from the four known type I gene families: 1) E2A, which encodes E12, E47, and ITF-1; 2) HEB, which in rat is homologous to REB-alpha and REB-beta ; 3) E2-2, corresponding to the rat and human ITF-2 proteins; and 4) the c-Myc-related multiprotein family. Systematic immunological screening assays eliminated E2A, E2-2, HEB, and c-Myc or its dimeric partner Max as candidate smE1 or smE2 binding factors (data not shown). However, as shown in Fig. 5, two c-Myc-related bHLH-ZIP proteins were identified within the smE1 binding complex. Specific antibodies against 43-kDa USF-1 and the related 44-kDa family member USF-2 reduced the amount of primary smE1 binding complex and formed a supershifted complex with electrophoretic mobility identical to that formed with SMC extract and a consensus USF binding site oligonucleotide probe. In vitro translation reactions programmed with full-length USF-1 and USF-2 expression constructs generated an smE1 binding complex with electrophoretic mobility identical to SMC-derived complexes (data not shown), again indicating that a major portion of smE1 binding activity derived from cultured SMCs is comprised of USF-containing complexes.


View larger version (114K):
[in this window]
[in a new window]
 
Fig. 5.   Supershift assay to screen for upstream stimulatory factors USF-1 and USF-2 in smE1 and smE2 binding complexes. SMC nuclear extracts were combined with radiolabeled oligonucleotides with smE1, smE2, or USF consensus binding site from adenovirus major later promoter (25) (positive control). Reactions were then mixed with antibodies specific to USF-1 alone (U1), USF-2 alone (U2), or a 1:1 mixture of both antibodies (1 + 2). Binding complexes were resolved as described in Fig. 3 legend. smE1 binding complex and USF consensus site were supershifted with each antibody in a pattern similar to that for SMC nuclear extract plus consensus USF binding site. In contrast, anti-USF antibodies did not significantly alter mobility of smE2 binding complexes. Although samples containing USF consensus oligonucleotide were electrophoresed alongside smE1- and smE2-containing samples, a shorter exposure of USF-containing lanes is shown for clarity.

In contrast to smE1, antibodies against USF-1, USF-2, or other bHLH factors had no effect on the mobility of the smE2 binding complex, providing further evidence for differential binding of SMC nuclear proteins to smE1 vs. smE2 probes. However, given the lack of any functional evidence that smE2 acted as an E-box in an SMC context (Fig. 2), that smE2-binding complex contained none of the previously described type I bHLH factors, and that the specificity of the interaction of smE2 with SMC nuclear proteins in competition assays was relatively low, identification of the smE2 binding factors was not pursued further for this study.

Cotransfection of USF-1, USF-2, or myogenin expression constructs with pCAT.271/native increased promoter activity in SMCs and non-SMC lines. Because results of gel-shift analyses with L6 myotube extracts demonstrated binding of myogenin to smE1 and smE2 (Fig. 3), we used 10T1/2 fibroblasts [which are permissive for transactivation of E-box-dependent skeletal muscle promoters (31, 33, 38)] to determine whether myogenin overexpression would transactivate the p271.CAT/native reporter. 10T1/2 fibroblasts were cotransfected with p271.CAT reporter plus an SV40-driven constitutive myogenin expression construct, then induced to differentiate and analyzed for CAT reporter activity, as described above. Our results demonstrated that myogenin induced an ~30-fold increase in CAT reporter activity in 10T1/2 fibroblasts (Fig. 6A). Moreover, mutation of smE1 or smE2 alone each reduced myogenin transactivation by one-half or more, indicating that transactivation by myogenin was E-box dependent.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of overexpression of myogenin or USF on transcriptional activity of wild-type and mutant pCAT.271/SMalpha A promoter-reporter constructs. A: effects of overexpression of myogenin on activity of wild-type and E-box mutant pCAT.271 constructs in a cell line permissive for myogenin transactivation; 3 µg of pCAT.271/native or 4 bp of smE1- or smE2-mutant (Table 1) reporter constructs were cotransfected into fibroblasts with no additional DNA (0), 1 µg of empty pEMSV [an SV40-driven constitutive expression vector (Vec)], or 1 µg of pEMSV/myogenin expression vector (Mg) with use of calcium phosphate. Fibroblasts were switched to differentiation medium 24 h after transfection, and CAT activity was determined 48 h later. All promoter activities are expressed as fold induction over native promoter, with pCAT.271/native = 1. B: effects of USF-1 or USF-2 overexpression on wild-type and E-box mutant SMalpha A promoter constructs in SMCs; 4 µg of pCAT.271/native or smE1 or smE2 mutants were cotransfected with 1 µg of cytomegalovirus (CMV)-driven constitutive expression vector alone (Vect) or expression plasmid containing coding region for human USF-1 (U1) or murine USF-2 (U2). All promoter activities are expressed as fold induction over native promoter, with pCAT.271/native = 1.

To determine whether USFs could transactivate the SMalpha A promoter, USF-1 or USF-2 expression constructs were cotransfected into SMCs with SMalpha A promoter-CAT reporter constructs containing the native vs. 4-bp smE1 or smE2 mutant E-boxes. Overexpression of USF-1 from a CMV promoter stimulated an approximately threefold increase in activity of p271.CAT/native (Fig. 6B); qualitatively equivalent results were observed using SV40-driven USF constitutive expression vectors (data not shown). Cotransfection of p271.CAT/native with USF-2 or a 1:1 mixture of USF-1 and USF-2 expression constructs also increased native promoter activity ~2.5-fold vs. native promoter plus empty coexpression vector. USF overexpression failed to transactivate a p271.CAT/E1m construct containing a 4-bp disrupting mutation at smE1 but did transactivate a p271.CAT/E2m reporter containing a 4-bp mutation at smE2, indicating that USF transactivation in SMCs was dependent on the smE1 element but did not require smE2. Loss of USF and myogenin transactivation also confirmed that, as we had predicted from published studies, the 4-bp mutations of smE1 and smE2 employed in this study completely disrupted functional interactions between bHLH factors and these E-box elements.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous deletion analyses of the rat SMalpha A promoter have demonstrated the presence of multiple positive and negative cis-acting regulatory elements that establish tissue-restricted expression of this gene in cultured cells (7, 28, 41). The present studies provide novel evidence indicating that two E-box motifs within the 5'-region of the SMalpha A promoter contribute to activity in skeletal myotubes and SMCs, although via distinct mechanisms and bHLH transcription factors. In L6 myotubes, smE1 and smE2 functioned as canonical E-boxes that were required for promoter activity. Moreover, smE1 and smE2 bound and were transactivated by the skeletal muscle-specific bHLH factor myogenin. In contrast, smE1 or smE2 alone was dispensable for promoter activity in cultured SMCs, although simultaneous mutation of smE1 and smE2 resulted in reduced promoter activity. In addition, in SMCs, smE1 but not smE2 appeared to function as a canonical E-box that bound the ubiquitously expressed bHLH-ZIP factor USF, which could transactivate the promoter.

The results of this study demonstrate that, similar to the previously described cardiac and skeletal alpha -actin genes (32, 38), SMalpha A promoter activity is in part controlled by E-box elements. As we reported previously, the -125 to +43 region of the SMalpha A promoter (which does not contain smE1 or smE2) is not active in skeletal myotubes, suggesting that essential promoter elements for skeletal muscle expression lay upstream of -125. This report identified paired E-box elements within the upstream region that bind myogenin-containing transcription factor complexes and regulate SMalpha A expression in skeletal muscle cells. This regulatory pattern matches several previous reports which showed that expression of skeletal and cardiac alpha -actins in skeletal muscle cells is dependent on paired E-box elements binding skeletal muscle lineage-specific bHLHs (16, 38, 43). Thus transient skeletal muscle expression of SMalpha A, as occurs during embryonic development, appears to share at least some of the same regulatory signals that modulate skeletal alpha -actin. Indeed, it is interesting to speculate that sequence conservation of smE2 and, to a lesser extent, smE1 across multiple mammalian SMalpha A promoters may be due to their requirement for paired E-boxes for SMalpha A promoter activity in skeletal muscle rather than an essential regulatory function in SMCs. We believe that this observation is potentially quite significant, since unlike most other targets of skeletal muscle-specific bHLHs, the SMalpha A gene is only transiently expressed within skeletal myotubes during early differentiation but is subsequently suppressed. As such, we believe that this gene may be particularly powerful for elucidating regulatory modules that differentially control the temporal and spatial expression of smooth muscle alpha -actin in skeletal vs. smooth muscle.

Earlier studies have established a model in which expression of tissue-specific genes during skeletal muscle differentiation is controlled primarily by MyoD, myogenin, and other bHLH factors that are specific for the muscle lineage and bind to E-box elements (1, 13, 32, 33); similar contributions of the bHLH factors dHAND and eHAND have been shown to be critical during cardiac development (18, 45). However, more recent studies have shown that, even in skeletal muscle, cell-specific gene regulation is not simply conferred by a single cell-specific transcription factor such as MyoD. Rather, it is dependent on a complex combination of interactions between multiple regulatory regions or modules and their transacting factors, including many factors that are not cell specific (14). In contrast to striated muscle, however, our evidence suggests that bHLH factors may not be the primary regulatory factor driving SMC-selective expression from the SMalpha A promoter. First, unlike myogenin, USF is ubiquitously expressed and alone cannot explain the high degree of cell selectivity exhibited by the SMalpha A gene. Second, the effects of the smE1 and smE2 double mutations on promoter activity in cultured SMCs were relatively modest compared with the effects in skeletal myotubes. However, our results provide clear evidence that the smE1-USF interaction has functional activity. Together, these data indicate that combinatorial interactions between multiple, non-tissue-specific factors may also contribute to smooth muscle-specific gene expression in a manner similar to many other cell types including cardiac and skeletal muscle (14).

An alternative interpretation of our data would be that USF contributes in other ways to the overall activity of this promoter rather than controlling tissue-specific gene expression per se. For example, USFs may modulate SMalpha A expression during different phenotypic states, as has been suggested for the MLC-2v gene (31) or in response to growth factors, e.g., TGF-beta (43). Consistent with this hypothesis, expression of c-Myc (a second bHLH-ZIP class factor) has been shown to be associated with downregulation of SMalpha A expression in SMCs during proliferation (3). As members of the c-Myc-related family, USFs can compete with c-Myc for similar DNA binding sites (2, 27, 46) and so may indirectly stimulate SMalpha A expression by inhibiting c-Myc-induced gene repression. It is also possible that activation of the SMalpha A promoter by USFs is essential in vivo but is dispensable in vitro, where there is compensatory promoter activation associated with phenotypic modulation that, in turn, masks a requirement for USF. Clearly, elucidation of the potential role of the smE1 (and smE2) element in modulation of SMalpha A expression in SMCs will be dependent on mutational studies in transgenic mice. Such studies have just recently been made possible by our identification of sufficient regions of the SMalpha A promoter necessary to drive expression of a LacZ reporter gene in vivo in a manner that recapitulates expression of the endogenous SMalpha A gene (27a).

Our initial observations (Fig. 2B) indicated that the presence of smE1 or smE2 alone was sufficient to maintain SMalpha A promoter activity in SMCs. Thus we had originally hypothesized that smE1 and smE2 represented redundant canonical E-box elements in SMCs and that binding of a similar bHLH factor complex to smE1 or smE2 was sufficient for maintaining promoter activity in SMCs. However, supershift and immunoblot analyses did not detect USFs, other c-Myc-related proteins, or ubiquitously expressed E2A, E2-2, and HEB-related type 1 bHLH factors within the smE2 binding complexes. Although not every possible tissue-restricted bHLH factor was exhaustively tested, the tissue-specific bHLH factors that have been identified previously are absent from mature arteries. Other candidates such as TAL1 and members of the HES family also have very stringent binding site specificities (5, 23, 49) and can be eliminated on the basis of lack of any effect of mutations in the 5'-CA or 3'-TG of smE2 in transfection assays (Fig. 2B). Moreover, given our current knowledge of the E-box/bHLH regulatory system, even a novel, smooth muscle-specific bHLH factor would be predicted to utilize one of the ubiquitously expressed type I factors as its dimerization partner. Thus it is more likely that the smE2 shift complex reflects binding of non-bHLH proteins to a cis element that overlaps the E-box region. One analogous example would be the E-box binding repressor protein ZEB (15), a zinc-finger protein with a binding site that overlaps an E-box motif within the IgH promoter. ZEB is displaced from the IgH promoter by E2A binding and appears to be a critical step in B cell-specific control of the immunoglobulin heavy chain promoter. Obviously, further studies are required to resolve the nature and identity of the smE2 binding factor(s) and the mechanism whereby it can compensate for abolition of the SMalpha A smE1 motif within an SMC context. Given the divergence of SMCs from striated muscle cells in the utilization of the bHLH/E-box regulatory pathway, these future studies may help define the complex interactions between USFs and other transcription factors that establish the unique smooth muscle phenotype.

In summary, the studies described here provide the first evidence for novel, E-box-dependent regulation of the SMalpha A gene in skeletal muscle cells as well as SMCs. Importantly, however, the mechanisms of regulation are very different in these two cell types. Further studies are required to elucidate the role of the SMalpha A E-box elements in regulation in vivo, during normal development, and in pathophysiological states characterized by alterations in SMalpha A expression in SMC (34, 35, 40) and non-smooth muscle cell types (4, 10, 25, 37, 41, 42).


    ACKNOWLEDGEMENTS

We thank Dr. Stephen Hauschka (University of Washington, Seattle, WA) for the kind gift of antibodies against E2-2, Dr. Michèle Sawadogo (M. D. Anderson Cancer Center, Houston, TX) for clones of the human USF-1 and murine USF-2, Dr. Stephen Konieczny (Purdue University, West Lafayette, IN) for the myogenin expression plasmids, and Dr. Paul DiCorleto (Cleveland Clinic, Cleveland, OH) for the rat endothelial cells.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants R01 HL-38854 and P01 HL-19242 awarded to G. K. Owens. A. D. Johnson was supported by NHLBI Training Grant T32-HL-07355; American Heart Association, Virginia Affiliate, fellowship VA-96-F-26; and NHLBI National Research Service Award F32-HL-09648-01.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. K. Owens, Dept. of Molecular Physiology and Biological Physics, University of Virginia, Box 449, Rm. 2-29 Jordan Hall, Charlottesville, VA 22908 (E-mail: gko{at}virginia.edu).

Received 25 January 1999; accepted in final form 29 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Apone, S., and S. D. Hauschka. Muscle gene E-box control elements. Evidence for quantitatively different transcriptional activities and the binding of distinct regulatory factors. J. Biol. Chem. 270: 21420-21427, 1995[Abstract/Free Full Text].

2.   Argenton, F., Y. Arava, A. Aronheim, and M. D. Walker. An activation domain of the helix-loop-helix transcription factor E2A shows cell type preference in vivo in microinjected zebra fish embryos. Mol. Cell. Biol. 16: 1714-1721, 1996[Abstract].

3.   Bennett, M. R., G. I. Evan, and A. C. Newby. Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-gamma , heparin, and cyclic nucleotide analogues and induces apoptosis. Circ. Res. 74: 525-536, 1994[Abstract].

4.   Beranek, J. T. alpha -Smooth muscle actin is not a specific and, consequently, a reliable marker for smooth muscle cells. Hum. Pathol. 24: 813-814, 1993[Medline].

5.   Blackwell, T. K., and H. Weintraub. Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science 250: 1104-1110, 1990[Medline].

6.   Blanar, M. A., P. H. Crossley, K. G. Peters, E. Steingrimsson, N. G. Copeland, N. A. Jenkins, G. R. Martin, and W. J. Rutter. Meso1, a basic helix-loop-helix protein involved in mammalian presomitic mesoderm development. Proc. Natl. Acad. Sci. USA 92: 5870-5874, 1995[Abstract/Free Full Text].

7.   Blank, R. S., T. C. McQuinn, K. C. Yin, M. M. Thompson, K. Takeyasu, R. J. Schwartz, and G. K. Owens. Elements of the smooth muscle alpha -actin promoter required in cis for transcriptional activation in smooth muscle. Evidence for cell type-specific regulation. J. Biol. Chem. 267: 984-989, 1992[Abstract/Free Full Text].

8.   Carter, R. S., P. Ordentlich, and T. Kadesch. Selective utilization of basic helix-loop-helix leucine zipper proteins at the immunoglobulin heavy-chain enhancer. Mol. Cell. Biol. 17: 18-23, 1997[Abstract].

9.   Cross, J. C., M. L. Flannery, M. A. Blanar, E. Steingrimsson, N. A. Jenkins, N. G. Copeland, W. J. Rutter, and Z. Werb. Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development. Development 121: 2513-2523, 1995[Abstract/Free Full Text].

10.   Desmoulière, A., L. Rubbia-Brandt, G. Grau, and G. Gabbiani. Heparin induces alpha -smooth muscle actin expression in cultured fibroblasts and in granulation tissue myofibroblasts. Lab. Invest. 67: 716-726, 1992[Medline].

11.   Dignam, J. D., P. L. Martin, B. S. Shastry, and R. G. Roeder. Eukaryotic gene transcription with purified components. Methods Enzymol. 101: 582-598, 1983[Medline].

12.   Edelman, E. R., M. Simons, M. G. Sirois, and R. D. Rosenberg. c-myc in vasculo-proliferative disease. Circ. Res. 76: 176-182, 1995[Abstract/Free Full Text].

13.   Edmondson, D. G., and E. N. Olson. Helix-loop-helix proteins as regulators of muscle-specific transcription. J. Biol. Chem. 268: 755-758, 1993[Free Full Text].

14.   Firulli, A. B., and E. N. Olson. Modular regulation of muscle gene transcription: a mechanism for muscle cell diversity. Trends Genet. 13: 364-369, 1997[Medline].

15.   Genetta, T., D. Ruezinsky, and T. Kadesch. Displacement of an E-box-binding repressor by basic helix-loop-helix proteins: implications for B-cell specificity of the immunoglobulin heavy-chain enhancer. Mol. Cell. Biol. 14: 6153-6163, 1994[Abstract].

16.   Gilmour, B. P., G. R. Fanger, C. Newton, S. M. Evans, and P. D. Gardner. Multiple binding sites for myogenic regulatory factors are required for expression of the acetylcholine receptor gamma -subunit gene. J. Biol. Chem. 266: 19871-19874, 1991[Abstract/Free Full Text].

17.   Hautmann, M. B., C. S. Madsen, and G. K. Owens. A transforming growth factor-beta (TGF-beta ) control element drives TFG-beta -induced stimulation of smooth muscle alpha -actin gene expression in concert with two CArG elements. J. Biol. Chem. 272: 10948-10956, 1997[Abstract/Free Full Text].

18.   Hollenberg, S. M., R. Sternglanz, P. F. Cheng, and H. Weintraub. Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system. Mol. Cell. Biol. 15: 3813-3822, 1995[Abstract].

19.   Horton, R. M. In vitro recombination on mutagenesis of DNA: SOEing together tailor-made genes. In: Methods in Molecular Biology. PCR Protocols, Current Methods, and Applications, edited by B. A. White. Totowa, NJ: Humana, 1993, vol. 15, p. 251-261.

20.   Huang, J., T. K. Blackwell, L. Kedes, and H. Weintraub. Differences between MyoD DNA binding and activation site requirements revealed by functional random sequence selection. Mol. Cell. Biol. 16: 3893-3900, 1996[Abstract].

21.   Hungerford, J. E., G. K. Owens, W. S. Argraves, and C. D. Little. Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers. Dev. Biol. 178: 375-392, 1996[Medline].

22.   Inaba, T., T. Gotoda, S. Ishibashi, K. Harada, J. I. Ohsuga, K. Ohashi, Y. Yazaki, and N. Yamata. Transcription factor PU.1 mediates induction of c-fms in vascular smooth muscle cells: a mechanism for phenotypic change to phagocytic cells. Mol. Cell. Biol. 16: 2264-2273, 1996[Abstract].

23.   Ishibashi, M., Y. Sasai, S. Nakanishi, and R. Kageyama. Molecular characterization of HES-2, a mammalian helix-loop-helix factor structurally related to Drosophila hairy and Enhancer of split. Eur. J. Biochem. 215: 645-652, 1993[Abstract].

24.   Kemp, P. R., J. C. Metcalfe, and D. J. Grainger. ID---a dominant negative regulator of skeletal muscle differentiation---is not involved in maturation or differentiation of vascular smooth muscle cells. FEBS Lett. 368: 81-86, 1995[Medline].

25.   Kim, J.-H., P. R. Bushel, and C. C. Kumar. Smooth muscle alpha -actin promoter activity is induced by serum stimulation of fibroblast cells. Biochem. Biophys. Res. Commun. 190: 1115-1121, 1993[Medline].

26.   Luo, X., and M. Sawadogo. Functional domains of the transcription factor USF2: atypical nuclear localization signals and context-dependent transcriptional activation domains. Mol. Cell. Biol. 16: 1367-1375, 1996[Abstract].

27.   Luo, X., and M. Sawadogo. Antiproliferative properties of the USF family of helix-loop-helix transcription factors. Proc. Natl. Acad. Sci. USA 93: 1308-1313, 1996[Abstract/Free Full Text].

27a.   Mack, C. P., and G. K. Owens. Regulation of SM alpha-actin expression in vivo is dependent upon CArG elements within the 5' and first intron promoter regions. Circ. Res. 84: 852-861, 1999[Abstract/Free Full Text].

28.   McNamara, C. A., M. M. Thompson, S. M. Vernon, R. T. Shimizu, R. S. Blank, and G. K. Owens. Nuclear proteins bind a cis-acting element in the smooth muscle alpha -actin promoter. Am. J. Physiol. 268 (Cell Physiol. 37): C1259-C1266, 1995[Abstract/Free Full Text].

29.   Muller, M. M., E. Schreiber, W. Schaffner, and P. Matthias. Rapid test for in vivo stability and DNA binding of mutated octamer binding proteins with "mini-extracts" prepared from transfected cells. Nucleic Acids Res. 17: 6420, 1989[Medline].

30.   Murre, C., G. Bain, M. A. van Dijk, I. Engel, B. A. Furnari, M. E. Massari, J. R. Matthews, M. W. Quong, R. R. Rivera, and M. H. Stuiver. Structure and function of helix-loop-helix proteins. Biochim. Biophys. Acta 1218: 129-135, 1994[Medline].

31.   Navankasattusas, S., M. Sawadogo, M. van Bilsen, C. V. Dang, and K. R. Chien. The basic helix-loop-helix protein upstream stimulating factor regulates the cardiac ventricular myosin light-chain 2 gene via independent cis regulatory elements. Mol. Cell. Biol. 14: 7331-7339, 1994[Abstract].

32.   Olson, E. N. Interplay between proliferation and differentiation within the myogenic lineage. Dev. Biol. 154: 261-272, 1992[Medline].

33.   Olson, E. N. Regulation of muscle transcription by the MyoD family. The heart of the matter. Circ. Res. 72: 1-6, 1993[Abstract].

34.   Owens, G. K. Regulation of differentiation of vascular smooth muscle cells. Physiol. Rev. 75: 487-517, 1995[Abstract/Free Full Text].

35.   Owens, G. K. Role of alterations in the differentiated state of smooth muscle cells in atherogenesis. In: Atherosclerosis and Coronary Artery Disease, edited by V. Fuster, R. Ross, and E. J. Topol. Philadelphia, PA: Lippincott-Raven, 1996, vol. 1, p. 401-420.

36.   Roberts, V. J., R. Steenbergen, and C. Murre. Localization of E2A mRNA expression in developing and adult rat tissues. Proc. Natl. Acad. Sci. USA 90: 7583-7587, 1993[Abstract/Free Full Text].

37.   Ronnov-Jessen, L., and O. W. Petersen. Induction of alpha -smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia. Lab. Invest. 68: 696-707, 1993[Medline].

38.   Sartorelli, V., M. Kurabayashi, and L. Kedes. Muscle-specific gene expression. A comparison of cardiac and skeletal muscle transcription strategies. Circ. Res. 72: 925-931, 1993[Medline].

39.   Sasai, Y., R. Kageyama, Y. Tagawa, R. Shigemoto, and S. Nakanishi. Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split. Genes Dev. 6: 2620-2634, 1992[Abstract].

40.   Schwartz, S. M., D. deBlois, and E. R. O'Brien. The intima. Soil for atherosclerosis and restenosis. Circ. Res. 77: 445-465, 1995[Free Full Text].

41.   Shimizu, R. T., R. S. Blank, R. Jervis, S. C. Lawrenz-Smith, and G. K. Owens. The smooth muscle alpha -actin gene promoter is differentially regulated in smooth muscle versus non-smooth muscle cells. J. Biol. Chem. 270: 7631-7643, 1995[Abstract/Free Full Text].

42.   Simonson, M. S., K. Walsh, C. C. Kumar, P. Bushel, and W. H. Herman. Two proximal CArG elements regulate SM alpha -actin promoter, a genetic marker of activated phenotype of mesangial cells. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F760-F769, 1995[Abstract/Free Full Text].

43.   Skerjanc, I. S., and M. W. McBurney. The E box is essential for activity of the cardiac actin promoter in skeletal but not in cardiac muscle. Dev. Biol. 163: 125-132, 1994[Medline].

44.   Solway, J., J. Seltzer, F. F. Samaha, S. Kim, L. E. Alger, Q. Niu, E. E. Morrisey, H. S. Ip, and M. S. Parmacek. Structure and expression of a smooth muscle cell-specific gene, SM22alpha . J. Biol. Chem. 270: 13460-13469, 1995[Abstract/Free Full Text].

45.   Srivastava, D., P. Cserjesi, and E. N. Olson. A subclass of bHLH proteins required for cardiac morphogenesis. Science 270: 1995-1999, 1995[Abstract].

46.   Viollet, B., A. M. Lefrancois-Martinez, A. Henrion, A. Kahn, M. Raymondjean, and A. Martinez. Immunochemical characterization and transacting properties of upstream stimulatory factor isoforms. J. Biol. Chem. 271: 1405-1415, 1996[Abstract/Free Full Text].

48.   Wolf, C., C. Thisse, C. Stoetzel, B. Thisse, P. Gerlinger, and F. Perrin-Schmitt. The M-twist gene of Mus is expressed in subsets of mesodermal cells and is closely related to the Xenopus X-twi and the Drosophila twist genes. Dev. Biol. 143: 363-373, 1991[Medline].

49.   Yoon, S. O., and D. M. Chikaraishi. Isolation of two E-box binding factors that interact with the rat tyrosine hydroxylase enhancer. J. Biol. Chem. 269: 18453-18462, 1994[Abstract/Free Full Text].


Am J Physiol Cell Physiol 276(6):C1420-C1431
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society