Heavy Chain Promoter Activity Research Articles

Type IIx myosin heavy chain promoter activity during hindlimb immobilization

Myosin heavy chain (MyHC) is an important skeletal muscle contractile protein that is often regulated at the transcriptional level. During atrophy, fast IIx MyHC mRNA levels are increased 35 fold in young, predominantly slow-twitch soleus muscle. The purpose of this study was to determine if the 5′-flanking region of the rat MyHC IIx gene contains an atrophy-response element. 1007 base pairs of the 5′-flanking region (−1 kb promoter) of the rat MyHC IIx gene were cloned, and deletion mutations were made. Intramuscular injections of plasmid DNA containing the firefly luciferase gene under control of the rat MyHC IIx promoter were made into the soleus of 2-mo-old rats undergoing normal cage activity or 6 days of hindlimb immobilization (HI). Aliquots of soleus muscle homogenates were used for firefly luciferase activity and plasmid DNA isolation for normalization (using real-time PCR with primers and probe designed against the firefly luciferase gene). The −1 and −0.889 kb promoter activities were 1.46-fold and 1.3-fold higher, respectively, in HI soleus compared to control. Deletion to −0.754 and −0.617 kb decreased promoter activity 70% and 30%, respectively, in HI soleus compared to control. In summary, the −1 kb MyHC IIx promoter is responsive to HI, and deletional analyses suggest that the MyHC IIx promoter potentially contains an atrophy-response element between −0.889 and −0.754 kb. Sponsored by the National Institutes of Health: 5F32AR051640-02 (RAS).

CHF1/Hey2 suppresses SM-MHC promoter activity through an interaction with GATA-6

The bHLH transcription factor CHF1/Hey2 has been previously shown to regulate neointimal formation after vascular injury, but the mechanisms have not been fully elucidated. The zinc-finger protein GATA-6 has also been shown to regulate vascular smooth-muscle phenotype through regulation of smooth-muscle contractile protein gene expression. To address the potential mechanisms by which CHF1/Hey2 regulates vascular smooth-muscle phenotype switching, we investigated the effect of CHF1/Hey2 on GATA-6-dependent smooth-muscle myosin heavy chain promoter activity. When cotransfected into NIH3T3 cells, CHF1/Hey2 reduced GATA-6-dependent activation of the promoter by 90%. Exogenous p300 was not sufficient to overcome this repression effect, demonstrating that the inhibitor effect did not involve coactivation by p300. Coimmunoprecipitation studies demonstrated that CHF1/Hey2 interacts directly with GATA-6. Mutational analysis demonstrated that the bHLH domain is required for transcriptional repression. Our findings highlight an important transcriptional mechanism by which CHF1/Hey2 may affect smooth-muscle cell phenotype.

A G/C element mediates repression of the SM22alpha promoter within phenotypically modulated smooth muscle cells in experimental atherosclerosis.

A hallmark of smooth muscle cell (SMC) phenotypic switching in atherosclerotic lesions is suppression of SMC differentiation marker gene expression. Yet little is known regarding the molecular mechanisms that control this process. Here we show that transcription of the SMC differentiation marker gene SM22alpha is reduced in atherosclerotic lesions and identify a cis regulatory element in the SM22alpha promoter required for this process. Transgenic mice carrying the SM22alpha promoter-beta-galactosidase (beta-gal) reporter transgene were crossed to apolipoprotein E (ApoE)-/- mice. Cells of the fibrous cap, intima, and underlying media showed complete loss of beta-gal activity in advanced atherosclerotic lesions. Of major significance, mutation of a G/C-rich cis element in the SM22alpha promoter prevented the decrease in SM22alpha promoter-beta-gal reporter transgene expression, including in cells that compose the fibrous cap of the lesion and in medial cells in proximity to the lesion. To begin to assess mechanisms whereby the G/C repressor element mediates suppression of SM22alpha in atherosclerosis, we tested the hypothesis that effects may be mediated by platelet-derived growth factor (PDGF)-BB-induced increases in the G/C binding transcription factor Sp1. Consistent with this hypothesis, results of studies in cultured SMCs showed that: (1) PDGF-BB increased expression of Sp1; (2) PDGF-BB and Sp1 profoundly suppressed SM22alpha promoter activity as well as smooth muscle myosin heavy chain promoter activity through mechanisms that were at least partially dependent on the G/C cis element; and (3) a short interfering RNA to Sp1 increased basal expression and attenuated PDGF-BB induced suppression of SM22alpha. Together, these results support a model whereby a G/C repressor element within the SM22alpha promoter mediates transcriptional repression of this gene within phenotypically modulated SMCs in experimental atherosclerosis and provide indirect evidence implicating PDGF-BB and Sp1 as possible mediators of these effects.

Phosphatase 2 A represses the human alpha myosin heavy chain promoter activity

Phosphatases are an important component of the signal transduction pathway. Recently, various studies have shown that phophatase (e.g. Map Kinase Phosphatases 1 and 2 (MKP 1 and 2), calcineurin, PP1 and PP2A) levels and/or activity are up-regulated in the failing heart. These various phophatases are implicated in a variety of cellular and molecular processes. Heart failure is also characterized by changes in the expression levels of many of the components of the contractile apparatus. Of the changes that are observed in failing hearts, increases in b-MyHC, skeletal a-actin, and ANF, with coordinate decreases in a-MyHC and SRCA2a, termed fetal gene induction, are perhaps the most well-known. Changes in MyHC are likely to play an important role in the contractile dysfunction observed in the failing heart. Recent experiments have shown that in single cell experiments, cardiac myocytes expressing only 12% of their MyHC as alpha have 52% greater power output than those expressing only beta. More recently, findings that a mutation in the aMyHC gene can cause either hypertrophic or dilated cardiomyopathies in humans, emphasize the importance of the regulation of aMyHC mRNA and protein expression that is observed in the human failing heart. Recent findings from our laboratory suggest that PP2A mRNA levels are increased in the human failing heart. In order to understand the significance of this increase we sought to determine the regulation of the fetal gene program in response to PP2A. At low concentrations Okadaic Acid is a specific inhibitor of PP2A. Here we report that aMyHC promoter activity is up-regulated in Okadaic Acid-treated neonate rat ventricular myocytes (NRVMs) and over expression of a wild type PP2A construct results in repression of aMyHC promoter activity. Moreover, over expression of a dominant negative PP2A construct dramatically up-regulates aMyHC promoter activity. Our results suggest that PP2A plays an important role in mediating aMyHC expression levels, and that its up-regulation in the failing heart is likely to have a dramatic effect on the regulation of the fetal gene program. Future studies are needed to determine other proteins involved in the PP2A regulation of the fetal gene program.

The Inhibitory Effect of Trilinolein on Norepinephrine-Induced β-Myosin Heavy Chain Promoter Activity, Reactive Oxygen Species Generation, and Extracellular Signal-Regulated Kinase Phosphorylation in Neonatal Rat Cardiomyocytes

The myocardial protective effects of trilinolein, isolated from the traditional Chinese herb Sanchi (Panax notoginseng), are thought to be related to its antioxidant activity. However, the intracellular mechanism underlying the protective effect of trilinolein in the heart remains unclear. In the present study, we investigated the effect of trilinolein on norepinephrine (NE)-induced protein synthesis in cardiomyocytes. Cultured neonatal rat cardiomyocytes were stimulated with NE, then protein content, [³H]-leucine incorporation, and β-myosin heavy chain (β-MyHC) promoter activity were examined. The effect of trilinolein on NE-induced intracellular reactive oxygen species (ROS) generation was measured with a redox- sensitive fluorescent dye (2′,7′-dichlorofluorescin diacetate) and extracellular signal-regulated kinase (ERK) phosphorylation by Western blotting. Trilinolein inhibited NE-increased protein synthesis, β-MyHC promoter activity, and intracellular ROS. Both trilinolein and the antioxidant, N-acetyl-cysteine, decreased NE- and H₂O₂-induced protein synthesis, β-MyHC promoter activity, and ERK phosphorylation. These data indicate that trilinolein inhibits NE-induced protein synthesis via attenuation of ROS generation in cardiomyocytes.

The inhibitory effect of trilinolein on norepinephrine-induced beta-myosin heavy chain promoter activity, reactive oxygen species generation, and extracellular signal-regulated kinase phosphorylation in neonatal rat cardiomyocytes.

The myocardial protective effects of trilinolein, isolated from the traditional Chinese herb Sanchi (Panax notoginseng), are thought to be related to its antioxidant activity. However, the intracellular mechanism underlying the protective effect of trilinolein in the heart remains unclear. In the present study, we investigated the effect of trilinolein on norepinephrine (NE)-induced protein synthesis in cardiomyocytes. Cultured neonatal rat cardiomyocytes were stimulated with NE, then protein content, [(3)H]-leucine incorporation, and beta-myosin heavy chain (beta-MyHC) promoter activity were examined. The effect of trilinolein on NE-induced intracellular reactive oxygen species (ROS) generation was measured with a redox- sensitive fluorescent dye (2',7'-dichlorofluorescin diacetate) and extracellular signal-regulated kinase (ERK) phosphorylation by Western blotting. Trilinolein inhibited NE-increased protein synthesis, beta-MyHC promoter activity, and intracellular ROS. Both trilinolein and the antioxidant, N-acetyl-cysteine, decreased NE- and H(2)O(2)-induced protein synthesis, beta-MyHC promoter activity, and ERK phosphorylation. These data indicate that trilinolein inhibits NE-induced protein synthesis via attenuation of ROS generation in cardiomyocytes.

Inhibitory effect of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy

The myocardial protective effects of trilinolein, isolated from the Chinese herb Sanchi ( Panax notoginseng), may be related to its antioxidant effects. In the present study, we investigated the effects of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy. Cultured neonatal rat cardiomyocytes were stimulated with angiotensin II, [ 3H]leucine incorporation and the β-myosin heavy chain promoter activity were examined. We also examined the effects of trilinolein on angiotensin II-induced intracellular reactive oxygen species generation. Trilinolein significantly inhibited angiotensin II-increased protein synthesis, β-myosin heavy chain promoter activity, and intracellular reactive oxygen species generation. Antioxidant N-acetylcysteine also decreased angiotensin II-increased protein synthesis and β-myosin heavy chain promoter activity. Furthermore, trilinolein and N-acetylcysteine decreased angiotensin II- or hydrogen peroxide (H 2O 2)-activated mitogen-activated protein kinases (MAPKs) phosphorylation, and activator protein-1 (AP-1)- [or nuclear factor-κB (NF-κB)]-reporter activities. These data indicate that trilinolein inhibits angiotensin II-induced cardiomyocyte hypertrophy and β-myosin heavy chain promoter activity via attenuation of reactive oxygen species generation.

Role of USF1 phosphorylation on cardiac α-myosin heavy chain promoter activity

Contractile activity of the cardiac myocyte is required for maintaining cell mass and phenotype, including expression of the cardiac-specific alpha-myosin heavy chain (alpha-MHC) gene. An E-box hemodynamic response element (HME) located at position -47 within the alpha-MHC promoter is both necessary and sufficient to confer contractile responsiveness to the gene and has been shown to bind upstream stimulatory factor-1 (USF1). When studied in spontaneously contracting cardiac myocytes, there is enhanced binding of USF1 to the HME compared with quiescent cells, which correlates with a threefold increase in alpha-MHC promoter activity. A molecular mechanism by which contractile function modulates alpha-MHC transcriptional activity may involve signaling via phosphorylation of USF1. The present studies showed that purified rat USF1 was phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) but not casein kinase II. Phosphorylated USF1 by either PKC or PKA had increased DNA binding activity to the HME. PKC-mediated phosphorylation also leads to the formation of USF1 multimers as assessed by gel shift assay. Analysis of in vivo phosphorylated nuclear proteins from cultured ventricular myocytes showed that USF1 was phosphorylated, and resolution by two-dimensional gel electrophoresis identified at least two distinct phosphorylated USF1 molecules. These results suggest that endogenous kinases can covalently modify USF1 and provide a potential molecular mechanism by which the contractile stimulus mediates changes in myocyte gene transcription.

Transcriptional regulation of the major histocompatibility complex (MHC) class I heavy chain, TAP1 and LMP2 genes by the human papillomavirus (HPV) type 6b, 16 and 18 E7 oncoproteins.

We have examined the possibility that the E7 proteins of the high-risk human papillomavirus (HPV) type 16 and 18 and the oncogenic adenovirus (Ad) type 12 E1A protein share the ability to down-regulate the expression of components of the antigen processing and presentation pathway, as a common strategy in the evasion of immune surveillance during the induction of cell transformation. Expression of the HPV 18 E7 oncoprotein, like Ad 12 E1A, resulted in repression of the major histocompatibility complex (MHC) class I heavy chain promoter, as well as repression of a bidirectional promoter that regulates expression of the genes encoding the transporter associated with antigen processing subunit 1 (TAP1) and a proteasome subunit, low molecular weight protein 2 (LMP2). HPV 16 E7 also caused a reduction in class I heavy chain promoter activity, however it did not have any significant effect on the activity of the bidirectional promoter. Interestingly, expression of the low-risk HPV 6b E7 protein resulted in an increase in MHC class I heavy chain promoter activity, while repressing the TAP1/LMP2 promoter. Interference with the class I pathway could also explain the ability of low-risk HPVs in inducing benign lesions.

Transcriptional Coactivator p300 Stimulates Cell Type-specific Gene Expression in Cardiac Myocytes

Terminal differentiation is characterized by cell cycle arrest and the expression of cell type-specific genes. Previous work has suggested that the p300 family of transcriptional coactivators plays an important role in preventing the re-initiation of DNA synthesis in terminally differentiated cardiac myocytes. In this study, we investigated whether p300 proteins are also involved in the transcriptional activation of cell type-specific genes in these cells. Since p300 function can be abrogated through direct binding by the adenovirus E1A protein, we overexpressed E1A in cardiac myocytes using recombinant adenoviral vectors. The expression of transfected reporter genes driven by alpha- or beta-myosin heavy chain promoters was markedly diminished by expression of the 12 S E1A protein. In contrast, the activity of a promoter derived from the ubiquitously expressed beta-actin gene was affected only modestly. While an E1A mutant unable to bind members of the retinoblastoma family of pocket proteins decreased the activity of alpha- and beta-myosin heavy chain promoters to nearly the same extent as wild type 12 S E1A, transcriptional repression by a mutant defective for p300 binding was severely impaired. Furthermore, overexpression of p300 and, to an even greater extent, p300del33, a mutant lacking residues required for binding by E1A, relieved E1A's repression of beta-myosin heavy chain promoter activity while having no effect on the activity of the beta-actin promoter. Thus, E1A's transcriptional repression of cell type-specific genes in cardiac myocytes is mediated through its binding of p300 proteins, and these proteins appear to be involved in maintaining both cell type-specific gene expression and cell cycle arrest in cardiac myocytes.

Thyroid hormone regulation of alpha-myosin heavy chain promoter activity assessed by invivo DNA transfer in rat heart

We have studied the thyroid-hormone responsiveness of the alpha-myosin heavy chain (MHC) gene in vivo by directly injecting an expression vector containing the alpha-MHC 5' regulatory sequences (-613 to +421 base pairs) into the rat heart. In the expression vector pAM1Luc the alpha-MHC promoter elements direct the synthesis of firefly luciferase. Although thyroxine administration of both euthyroid and thyroidectomized rats for 5 days increased alpha-MHC promoter activity, the pAM1Luc gene construct did not mimic expression of the endogenous gene. These studies of direct gene transfer into mammalian myocardium suggest that additional cis-acting elements necessary for the in vivo response to thyroid hormone reside outside the -613 to +421 region.