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While it is well established that phenotypic modulation of vascular smooth muscle cells (VSMCs) contributes to the development and progression of vascular lesions, little is known regarding the molecular mechanisms of phenotypic modulation in vivo. Here we show that vascular injury reduces transcription of VSMC differentiation marker genes, and we identify cis regulatory elements that may mediate this decrease. Using a carotid wire-injury model in mice carrying transgenes for smooth muscle alpha-actin, smooth muscle myosin heavy chain, or a SM22alpha promoter-beta-gal reporter, we collected arteries 7 and 14 days after injury and assessed changes in endogenous protein and mRNA levels and in beta-gal activity. Endogenous levels for all markers were decreased 7 days after injury and returned to nearly control levels by 14 days. beta-gal staining in all lines followed a similar pattern, suggesting that transcriptional downregulation contributed to the injury-induced decreases. To begin to dissect this response, we mutated a putative G/C-rich repressor in the SM22alpha promoter transgene and found that this mutation significantly attenuated injury-induced downregulation. Hence, transcriptional downregulation contributes to injury-induced decreases in VSMC differentiation markers, an effect that may be partially mediated through a G/C-rich repressor element.

The smooth muscle myosin heavy chain (SM-MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM-MHC gene. Here we report the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle-specific expression in transgenic mice. Transgenic mice harboring an SM-MHC-lacZ reporter construct containing approximately 16 kb of the SM-MHC genomic region from -4.2 to + 11.6 kb (within the first intron) expressed the lacZ transgene in all smooth muscle tissue types. The inclusion of the intronic sequence was required for transgene expression, since 4.2 kb of the 5'-flanking region alone was not sufficient for expression. In the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, in airway smooth muscle of the trachea and bronchi, and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters, and bladder. During development, transgene expression was first detected in airway SMCs at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5. Of interest, expression of the SM-MHC transgene was markedly reduced or absent in some SMC tissues, including the pulmonary circulation. Moreover, the transgene exhibited a heterogeneous pattern between individual SMCs within a given tissue, suggesting the possibility of the existence of different SM-MHC gene regulatory programs between SMC subpopulations and/or of episodic rather than continuous expression of the SM-MHC gene. To our knowledge, results of these studies are the first to identify a promoter region that confers complete SMC specificity in vivo, thus providing a system with which to define SMC-specific transcriptional regulatory mechanisms and to design vectors for SMC-specific gene targeting.

We have previously demonstrated that both CC(A/T-rich)6GG (CArG) elements A and B of the smooth muscle (SM) alpha-actin promoter are required for smooth muscle cell (SMC)-specific expression and angiotensin II (AII)-induced stimulation. Moreover, results provided evidence that AII responsiveness of SM alpha-actin was at least partially dependent on modulation of serum response factor (SRF) binding to the SM alpha-actin CArGs by the homeodomain containing protein, MHox. The goal of the present study was to investigate whether the degeneracy of the SM alpha-actin CArGs (both contain a Gua or Cyt substitution in their A/T-rich center) and their reduced SRF binding activity as compared with c-fos serum response element (SRE) is important for conferring cell type-specific expression and AII responsiveness. Transient transfection assays using SM alpha-actin reporter gene constructs in which the endogenous SM alpha-actin CArGs were replaced by c-fos SREs demonstrated the following: 1) relaxation of cell-specific expression, 2) a 50% reduction in AII responsiveness, and 3) reduced ability to be transactivated by MHox. In addition, we also showed that the position of the SM alpha-actin CArGs was important in that interchanging them abolished both basal and AII-induced activities. Taken together, these results suggest that the reduced SRF binding activities of the SM alpha-actin CArGs and CArG positional context contribute to SMC-specific expression of SM alpha-actin as well as maximal AII responsiveness.

We have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides -1321 and -1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997) J. Biol. Chem. 272, 6332-6340). In this study, to better understand the functional role of these three cis elements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elements in vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions.

The goal of the present study was to determine the molecular mechanism whereby transforming growth factor beta (TGFbeta) increases smooth muscle (SM) alpha-actin expression. Confluent, growth-arrested rat aortic smooth muscle cells (SMC) were transiently transfected with various SM alpha-actin promoter/chloramphenicol acetyltransferase deletion mutants and stimulated with TGFbeta (2.5 ng/ml). Results demonstrated that the first 125 base pairs of the SM alpha-actin promoter were sufficient to confer TGFbeta responsiveness. Three cis elements were shown to be required for TGFbeta inducibility: two highly conserved CArG boxes, designated A (-62) and B (-112) and a novel TGFbeta control element (TCE) (-42). Mutation of any one of these elements completely abolished TGFbeta-induced reporter activity. Results of electrophoretic mobility shift assays demonstrated that nuclear extracts from TGFbeta-treated SMC enhanced binding activity of serum response factor to the CArG elements and binding of an as yet unidentified factor to the TCE. Northern analysis showed that TGFbeta also stimulated transcription of two other SM (SM myosin heavy chain) differentiation marker genes, SM myosin heavy chain and h1 calponin, whose promoters also contained a TCE-like element. In summary, we identified a TGFbeta response element in the SM alpha-actin promoter that may contribute to coordinate regulation of expression of multiple cell-type specific proteins during SMC differentiation.

To identify cis- and trans-acting factors that regulate smooth muscle-specific gene expression, we studied the smooth muscle myosin heavy chain gene, a rigorous marker of differentiated smooth muscle. A comparison of smooth muscle myosin heavy chain promoter sequences from multiple species revealed the presence of a highly conserved 227-base pair domain (nucleotides -1321 to -1095 in rat). Results of a deletion analysis of a 4.3-kilobase pair segment of the rat promoter (nucleotides -4220 to +88) demonstrated that this domain was necessary for maximal transcriptional activity in smooth muscle cells. Gel-shift analysis and site-directed mutagenesis demonstrated that one true CArG and another CArG-like element contained within this domain were both recognized by the serum response factor and were both required for the positive activity attributable to this domain. Additional studies demonstrated that mutation of a GC-rich sequence within the 227-base pair conserved domain resulted in a nearly 100% increase in transcriptional activity. Gel-shift analysis showed that this GC-rich repressor element was recognized by both Sp1 and Sp3. These data demonstrate that transcriptional control of the smooth muscle myosin heavy chain gene is highly complex, involving both negative and positive regulatory elements, including CArG sequences found in the promoters of multiple smooth muscle differentiation marker genes.

REGULATION OF SMOOTH MUSCLE CELL (SMC) GROWTH: Accelerated growth of SMC is known to play an integral role in atherosclerotic lesion formation as well as post-angioplasty restenosis, and is a characteristic feature in arteries of hypertensive patients and animals. There has thus been extensive interest in defining both positive and negative regulators of SMC growth, and many factors have been identified that may play a role in this process. REGULATION OF DIFFERENTIATED SMC: Despite clear evidence that the differentiated state of the intimal SMC is altered in atherosclerotic disease, and that this is likely to play a key role in lesion development, relatively little is known about the mechanisms and factors that regulate the differentiated state of SMC. Extrinsic factors (local environmental cues) make an important contribution to the regulation of the differentiated state of the vascular SMC. Molecular mechanisms control the expression of genes encoding for proteins such as smooth muscle alpha-actin and smooth muscle myosin heavy chain that are selective or specific for SMC, and which are required for the SMC principal differentiated function, contraction.