Smooth Muscle-specific Genes Research Articles

Development of a smooth muscle-targeted cre recombinase mouse reveals novel insights regarding smooth muscle myosin heavy chain promoter regulation.

The use of genetically modified mice has been an important model system to study gene function in cardiovascular development and under pathophysiological conditions. Although conventional gene knockout studies have provided important insights into gene function in the cardiovascular system, they may be limited by upregulation of compensatory pathways and the inability to differentiate direct versus indirect functions in vivo. As a first step in developing systems that can target gene activation or inactivation specifically to smooth muscle cells (SMCs), we coupled the smooth muscle myosin heavy chain (SMMHC) promoter to the cre recombinase gene and generated transgenic mice that express cre in SMCs. In addition, we used these mice to address whether the heterogeneous staining observed in SMMHC-LacZ mice was due to subsets of SMCs that required different regulatory cassettes of the promoter or if it reflected episodic expression of the transgene. To address both the feasibility of SMC targeting and the apparent heterogeneous expression, we bred SMMHC-cre mice to indicator mice containing a cre-activated LacZ gene. Results showed high-level expression in SMCs at various embryonic time points and in adult tissues. Because breeding of SMMHC-cre mice to an indicator line provided an integration of cre activity over time, results of this study revealed that expression of the SMMHC promoter fragment more closely resembled the expression of the endogenous gene, both with respect to the onset of activation during development and uniformity of staining among individual cells within tissues. Overall, these mice will provide a powerful tool to researchers to study gene function in vascular development/disease by using cre/lox technology to direct smooth muscle-specific gene activation or inactivation in vivo.

Physiological Control of Smooth Muscle-specific Gene Expression through Regulated Nuclear Translocation of Serum Response Factor

Prolonged serum deprivation induces a structurally and functionally contractile phenotype in about 1/6 of cultured airway myocytes, which exhibit morphological elongation and accumulate abundant contractile apparatus-associated proteins. We tested the hypothesis that transcriptional activation of genes encoding these proteins accounts for their accumulation during this phenotypic transition by measuring the transcriptional activities of the murine SM22 and human smooth muscle myosin heavy chain promoters during transient transfection in subconfluent, serum fed or 7 day serum-deprived cultured canine tracheal smooth muscle cells. Contrary to our expectation, SM22 and smooth muscle myosin heavy chain promoter activities (but not viral murine sarcoma virus-long terminal repeat promoter activity) were decreased in long term serum-deprived myocytes by at least 8-fold. Because serum response factor (SRF) is a required transcriptional activator of these and other smooth muscle-specific promoters, we evaluated the expression and function of SRF in subconfluent and long term serum-deprived cells. Whole cell SRF mRNA and protein were maintained at high levels in serum-deprived myocytes, but SRF transcription-promoting activity, nuclear SRF binding to consensus CArG sequences, and nuclear SRF protein were reduced. Furthermore, immunocytochemistry revealed extranuclear redistribution of SRF in serum-deprived myocytes; nuclear localization of SRF was restored after serum refeeding. These results uncover a novel mechanism for physiological control of smooth muscle-specific gene expression through extranuclear redistribution of SRF and consequent down-regulation of its transcription-promoting activity.

Medial localization of mineralization-regulating proteins in association with Mönckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification.

Calcification of the media of peripheral arteries is referred to as Mönckeberg's sclerosis (MS) and occurs commonly in aged and diabetic individuals. Its pathogenesis is unknown, but its presence predicts risk of cardiovascular events and leg amputation in diabetic patients. Several studies have documented expression of bone-associated genes in association with intimal atherosclerotic calcification, leading to the suggestion that vascular calcification may be a regulated process with similarities to developmental osteogenesis. Therefore, we examined gene expression in vessels with MS to determine whether there was evidence for a regulated calcification process in the vessel media. In situ hybridization, immunohistochemistry, and semiquantitative reverse-transcription polymerase chain reaction were used to examine the expression of mineralization-regulating proteins in human peripheral arteries with and without MS. MS occurred in direct apposition to medial vascular smooth muscle cells (VSMCs) in the absence of macrophages or lipid. These VSMCs expressed the smooth muscle-specific gene SM22alpha and high levels of matrix Gla protein but little osteopontin mRNA. Compared with normal vessels, vessels with MS globally expressed lower levels of matrix Gla protein and osteonectin, whereas alkaline phosphatase, bone sialoprotein, bone Gla protein, and collagen II, all indicators of osteogenesis/chondrogenesis, were upregulated. Furthermore, VSMCs derived from MS lesions exhibited osteoblastic properties and mineralized in vitro. These data indicate that medial calcification in MS lesions is an active process potentially orchestrated by phenotypically modified VSMCs.

Characterization of an E-box-dependent cis element in the smooth muscle alpha-actin promoter.

Identification of the regulators of smooth muscle specific gene expression is critical for understanding smooth muscle cell (SMC) differentiation and the alterations in SMC phenotype seen in vascular diseases. Previous studies have identified that a 2-bp mutation in a conserved cis-acting element (TGTTTATC) in the promoter of the chicken smooth muscle (SM) alpha-actin gene abolished nuclear factor binding and decreased transcriptional activity of a 271-bp SM alpha-actin promoter fragment when transfected into rat aortic SMC. However, the promoter region containing this conserved sequence has negative cis regulatory activity when studied in homologous systems. The goal of the present studies was to further characterize the transcriptional activity of the rat SM alpha-actin promoter region between -224 and -236 that is conserved across mammals. DNAse I analysis and electrophoretic mobility shift assays demonstrated that SMC nuclear proteins bound an extended sequence (TGTTTATCCCCATAA). Transient transfection experiments of wild-type and mutant rat SM alpha-actin promoter-luciferase constructs into rat aortic SMC revealed that promoter activity was enhanced by mutations of specific nucleotides in the TGTTTATCCCCA region. Interestingly, the TGTTTATCCCCA element in the rat SM alpha-actin promoter is centered between 2 canonical E-boxes. Mutations of the flanking E-boxes abolished the enhancement in promoter activity seen with mutation of the TGTTTATCCCCA element alone. Thus studies provide evidence for a regulatory cassette in the rat SM alpha-actin promoter that regulates gene expression via combinatorial interactions between 2 E-boxes and a newly described TGTTTATCCCCA element.

Expression of Smooth Muscle Calponin in Synovial Sarcoma

Purpose. Histogenesis of synovial sarcoma remains controversial and reliable molecular markers for diagnosis are necessary. Expression of basic calponin, a smooth muscle differentiation-specific actin-binding protein, was studied in synovial sarcoma. Subjects and Methods. The basic calponin gene and the gene product were analyzed by reverse transcription PCR analysis (RT-PCR) and immunohistochemistry in 14 synovial sarcomas and a human synovial sarcoma cell line (HS-SY-II). Results and Discussion. Immunoreactivity for basic calponin was detected in the cytoplasm of 6 synovial sarcomas (43% positive). In the basic calponin-positive tumors and the HS-SY-II cells, expression for smooth muscle-specific genes, including basic calponin and SM22α , was detected by RT-PCR, suggesting a lineage relationship between synovial sarcoma cells and smooth muscle-like mesenchymal cells. Conclusions. A subset of synovial sarcomas expressing the basic calponin gene and the gene product were identified. The basic calponin may have potential utility as a novel molecular marker identifying certain synovial sarcomas.

Transcriptional regulation of smooth muscle contractile apparatus expression.

The transcriptional regulatory mechanisms that control gene expression during differentiation and contractile protein accumulation are becoming well understood in skeletal and cardiac muscle lineages. Current understanding of smooth muscle-specific gene transcription is much more limited, though recent studies have begun to shed light on this topic. In this review, we summarize some of the themes emerging from these studies and identify transcriptional regulatory elements common to several smooth muscle genes. These include potential binding sites for serum response factor, Sp1, AP2, Mhox, and YY1, as well as a potential transforming growth factor-beta control element. We speculate that it may be possible to manipulate smooth muscle-specific gene expression in asthma or pulmonary arterial hypertension as an eventual therapy.

Differentiated Phenotype of Smooth Muscle Cells Depends on Signaling Pathways through Insulin-like Growth Factors and Phosphatidylinositol 3-Kinase

Under conventional culture conditions, smooth muscle cells display their phenotypic modulation from a differentiated to a dedifferentiated state. Here, we established a primary culture system of smooth muscle cells maintaining a differentiated phenotype, as characterized by expression of smooth muscle-specific marker genes such as h-caldesmon and calponin, cell morphology, and ligand-induced contractility. Laminin retarded the progression of dedifferentiation of smooth muscle cells. Insulin-like growth factors (IGF-I and IGF-II) and insulin markedly prolonged the differentiated phenotype, with IGF-I being the more potent. In contrast, serum, epidermal growth factor, transforming growth factors, and platelet-derived growth factors potently induced dedifferentiation compared with angiotensin II, arginine-vasopressin, and basic fibroblast growth factor. Using the present culture system, we investigated signaling pathways regulating a phenotype of smooth muscle cells. In cultured cells, IGF-I specifically activated phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, protein kinase B, but not mitogen-activated protein kinases. Specific inhibitors of PI3-kinase (wortmannin and LY294002) induced dedifferentiation of smooth muscle cells even when they were cultured on laminin under IGF-I-stimulated conditions. The sole effect of laminin to retard the dedifferentiation was completely blocked by anti-IGF-I antibody, and laminin promoted the endogenous expression of IGF-I in cultured cells. The reduced promoter activity of the caldesmon gene induced by platelet-derived growth factor BB was overcome by the forced expression of the constitutive active form of PI3-kinase p110alpha catalytic subunit. These findings suggest that an IGF-I signaling pathway through PI3-kinase plays a critical role in maintaining a differentiated phenotype of smooth muscle cells.

Expression of the smooth muscle calponin gene in human osteosarcoma and its possible association with prognosis.

The basic calponin gene is a smooth muscle differentiation-specific gene that encodes an actin-binding protein involved in the regulation of smooth muscle contractility. We studied the expression of the calponin gene in 8 human osteosarcoma cell lines and 17 primary human osteosarcoma tissues by RT-PCR analysis. We also analyzed mRNA expression of smooth muscle-specific genes including SM22alpha, caldesmon and alpha-actin, and for neutral and acidic calponin isoforms. The genes were expressed at various levels by osteosarcoma cell lines and tissues of diverse histological subtypes. The basic calponin protein of an expected size was detected in osteosarcoma cell lines by immunoblot analysis and was localized by immunohistochemistry in the cytoplasm of the tumor cells in osteosarcoma tissues. Survival was found to be significantly increased in patients whose tumors exhibited basic calponin expression, compared with those with no expression. Alterations in the expression of other markers examined were not correlated with prognosis. Our results suggest that the basic calponin gene product may be a novel prognostic variable in patients with osteosarcoma.

Enhancement of Serum-response Factor-dependent Transcription and DNA Binding by the Architectural Transcription Factor HMG-I(Y)

The mechanisms by which HMG-I proteins regulate cell growth are unknown, and their effects on gene expression have only been partially elucidated. We explored the potential interaction between HMG-I proteins and serum-response factor (SRF), a member of the MADS-box family of transcription factors. In cotransfection experiments, HMG-I(Y) potentiated SRF-dependent activation (by more than 5-fold) of two distinct SRF-responsive promoters, c-fos and the smooth muscle-specific gene SM22alpha. This effect was also observed with a heterologous promoter containing multiple copies of the CC(A/T)6GG (CArG) box. HMG-I proteins bound specifically to the CArG boxes of c-fos and SM22alpha in gel mobility shift analysis and enhanced binding of SRF to these CArG boxes. By chelating peptide-immobilized metal affinity chromatography, we mapped the domain of HMG-I(Y) that interacts with SRF to amino acids 50-81, a region that does not bind specifically to DNA in electrophoretic mobility shift assays even though it includes the third AT-hook DNA-binding domain. Surprisingly, HMG-I(Y) mutants that failed to bind DNA still enhanced SRF binding to DNA and SRF-dependent transcription. In contrast, deletion of the HMG-I(Y) 50-81 domain that bound SRF prevented enhancement of transcription. To our knowledge, this is the first report of an HMG-I protein interacting with a MADS-box transcription factor. Our observations suggest that members of the HMG-I family play an important role in SRF-dependent transcription and that their effect is mediated primarily by a protein-protein interaction.

Evolutionarily conserved promoter region containing CArG*-like elements is crucial for smooth muscle myosin heavy chain gene expression.

In recent years, significant progress has been made toward understanding skeletal muscle development. However, the mechanisms that regulate smooth muscle development and differentiation are presently unknown. To better understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a highly specific marker of differentiated smooth muscle cells. The goal of the present study was to isolate and characterize the mouse SMHC gene promoter, since the mouse promoter would be particularly suited for in vivo promoter analyses in transgenic mice and would serve as a tool for targeting genes of interest into smooth muscle cells. We report here the isolation and characterization of the mouse SMHC promoter and its 5' flanking region. DNA sequence analysis of a 2.6-kb portion of the promoter identified several potential binding sites for known transcription factors. Transient transfection analysis of promoter deletion constructs in primary cultures of smooth muscle cells showed that the region between -1208 and -1050 bp is critical for maximal SMHC promoter activity. A comparison of SMHC promoter sequences from mouse, rat, and rabbit revealed the presence of a highly conserved region located between -967 and -1208 bp. This region includes three CArG/CArG*-like elements, two SP-1 binding sites, a NF-1-like element, an Nkx2-5 binding site, and an Elk-1 binding site. Gel mobility shift assay and DNase I footprinting analyses show that all three CArG/CArG*-like elements can form DNA-protein complexes with nuclear extract from vascular smooth muscle cells. Protein binding to the CArG* elements can be competed out by either serum response element or by an authentic CArG element from the cardiac alpha-actin gene. Using a serum response factor (SRF) antibody, we demonstrate that SRF is part of the protein complex. In addition, we show that cotransfection with the SRF dominant-negative mutant expression vector abolishes SMHC promoter activity, suggesting that SRF protein plays a critical role in SMHC gene regulation.

The Developmentally Regulated Expression of Serum Response Factor Plays a Key Role in the Control of Smooth Muscle-Specific Genes

Serum response factor (SRF) is a MADS box transcription factor that has been shown to be important in the regulation of a variety of muscle-specific genes. We have previously shown SRF to be a major component of multiplecis/transinteractions found along the smooth muscle γ-actin (SMGA) promoter. In the studies reported here, we have further characterized the role of SRF in the regulation of the SMGA gene in the developing gizzard. EMSA analyses, using nuclear extracts derived from gizzards at various stages in development, showed that the SRF-containing complexes were not present early in gizzard smooth muscle development, but appeared as development progressed. We observed an increase in SRF protein and mRNA levels during gizzard development by Western and Northern blot analyses, with a large increase just preceding an increase in SMGA expression. Thus, changes in SRF DNA-binding activity were paralleled with increased SRF gene expression. Immunohistochemical analyses demonstrated a correspondence of SRF and SMGA expression in differentiating visceral smooth muscle cells (SMCs) during gizzard tissue development. This correspondence of SRF and SMGA expression was also observed in cultured smooth muscle mesenchyme induced to express differentiated gene productsin vitro.In gene transfer experiments with SMGA promoter–luciferase reporter gene constructs we observed four- to fivefold stronger SMGA promoter activity in differentiated SMCs relative to replicating visceral smooth muscle cells. Further, we demonstrate the ability of a dominant negative SRF mutant protein to specifically inhibit transcription of the SMGA promoter in visceral smooth muscle, directly linking SRF with the control of SMGA gene expression. Taken together, these data suggest that SRF plays a prominent role in the developmental regulation of the SMGA gene.

MEF2B Is a Component of a Smooth Muscle-specific Complex That Binds an A/T-rich Element Important for Smooth Muscle Myosin Heavy Chain Gene Expression

To understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a smooth muscle-specific marker. In this study, we demonstrate that the SMHC promoter region (-1594 to -1462 base pairs) containing the A/T-rich element can activate the heterologous thymidine kinase promoter in smooth muscle cells, but not in fibroblasts. Mutations of this A/T-rich element decreased SMHC promoter activity significantly. Both gel mobility shift assays and DNase I footprinting revealed that this region binds to specific protein complexes from smooth muscle nuclear extracts, whereas nuclear extracts from skeletal muscle and fibroblasts produced a different binding pattern. We also demonstrate that the protein complex obtained from smooth muscle nuclear extract reacts with MEF2B-specific antibody, but not with antibodies specific to MEF2A, MEF2C, or MEF2D, suggesting that only MEF2B protein binds to the A/T-rich element. Furthermore, MEF2B overexpression in smooth muscle cells up-regulated the SMHC promoter, suggesting that MEF2B is important for SMHC gene regulation. This is the first report demonstrating a role for MEF2 factors in smooth muscle-specific gene expression.

Transfection with c-HarasEJModulatesα-actin andα1B-adrenoceptor Gene Expression in Vascular Smooth Muscle Cells

Co-ordinate down-regulation of smooth muscle-specific genes and acquisition of unregulated proliferative characteristics have been proposed as hallmarks of the atherosclerotic process. In the present study, we have evaluated this reciprocal relationship by examining the impact of c-Ha-rasEJoncogene transfection onα-smooth muscle (SM) actin andα1B-adrenoceptor (ADR) gene expression in vascular (aortic) smooth muscle cells (SMCs). c-Ha-rasEJtransfection of SMCs by lipofection (LF-1) was associated with enhanced DNA synthetic rates relative to vector controls and a significant reduction inα-SM actin andβ/γ-actin mRNAs. Incubation ofras- andneo-LF-1 SMCs in a restrictive serum concentration (0.1%) for 72 h inhibited DNA synthesis in both cell types, but differentially influenced the pattern ofα-actin gene expression. Whileneo-LF-1 cells incubated in 0.1% exhibited increasedα-SM actin mRNA levels relative to 10% serum, slight decreases inα-SM actin were observed inras-LF-1 cells under the same conditions. Cyclical stretch of randomly cycling cells, seeded on a flexible elastin substrate at a rate of 100 cycles/min for 72 h, did not significantly influence the pattern ofα-SM orβ/γ-actin mRNA expression inneo-LF-1 orras-LF-1 cells. Steady-state mRNA levels ofα1B-ADR were higher inras-LF-1 SMCs relative toneo-LF-1 cells, and stretch increasedα1B-ADR mRNA levels inneo-LF-1, but notras-LF-1 cells. Stretch inhibited [3H]thymidine incorporation into DNA in bothneo- andras-LF-1 cells relative to unstretched counterparts. These results demonstrate that c-Ha-rasEJtransfection is associated with alterations in the expression of genes associated with muscle-specific functions in vascular SMCs and implicate c-Ha-rasin the regulation of phenotypic expression in SMCs.

Expression of the Smooth Muscle Myosin Heavy Chain Gene Is Regulated by a Negative-acting GC-rich Element Located between Two Positive-acting Serum Response Factor-binding Elements

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.

Expression of the SM22alpha promoter in transgenic mice provides evidence for distinct transcriptional regulatory programs in vascular and visceral smooth muscle cells.

SM22alpha is a putative calcium-binding protein that is expressed in cardiac, smooth, and skeletal muscle lineages during mouse embryogenesis and in adult smooth muscle cells (SMC). To define the mechanisms that regulate smooth muscle-specific gene transcription, we isolated the SM22alpha gene and analyzed its 5'-flanking region for elements that direct smooth muscle expression in transgenic mice. Using a series of promoter deletions, a region of the SM22alpha promoter containing 445 base pairs of 5'-flanking sequence was found to be sufficient to direct the specific expression of a lacZ transgene in mouse embryos in the vascular smooth, cardiac, and skeletal muscle lineages in a temporospatial pattern similar to the endogenous SM22alpha gene. However, in contrast to the endogenous gene, transgene expression was not detected in venous, nor visceral SMCs. This SM22alpha-lacZ transgene was therefore able to distinguish between the transcriptional regulatory programs that control gene expression in vascular and visceral SMCs and revealed heretofore unrecognized differences between these SMC types. These results suggest that distinct transcriptional regulation programs control muscle gene expression in vascular and visceral SMCs.

Complete Nucleotide Sequence, Structural Organization, and an Alternatively Spliced Exon of Mouseh1-Calponin Gene

From a CC1.2 embryonic stem cell genomic library, we isolated and sequenced a 10.4-kb DNA segment (GenBank/EMBL Data Bank accession number L49022) containing the entire gene encoding mouseh1-calponin, an actin-associated smooth muscle-specific protein and a potential modulator of contraction. Sequence data revealed that there are seven exons and six introns in theh1-calponin gene. Determined by primer extension mapping of the RNA transcripts, the transcription ofh1-calponin gene initiates at the same site in stomach, urinary bladder and pregnant uterus smooth muscles. The genomic organization suggests that the previously identified α- and β-calponin isoforms are produced by splicing of exon 7 at two alternative acceptor sites. Isolation and structural characterization of theh1-calponin gene provides information to further investigate the expression regulation of this smooth muscle-specific gene.

The progression associated antigen MUC18

The cell surface glycoprotein MUC18 was originally identified as a progression associated antigen in melanoma. MUC18 is expressed most strongly on metastatic lesions and advanced primary tumours and is only rarely detected in benign lesions. cDNA cloning revealed MUC18 to be a novel member of the immunoglobulin superfamily with sequence similarity to a number of cell adhesion molecules. Cloning of both the human and mouse MUC18 genes indicate that their predicted protein structures are very similar with an overall amino acid identity of 75%. Like its human counterpart, murine MUC18 is also expressed by transformed melanocytes. Analysis of the promoter region of the human gene has provided evidence for regulatory elements found in smooth muscle specific genes and in both human and mouse: this is the normal site of MUC18 expression. The presence of putative binding sites for the transcriptional factors AP-1, AP-2 and CREB, suggest that MUC18 gene expression can also be modulated by external factors.