Differentiation of smooth muscle cells is accompanied by the transcriptional activation of an array of muscle-specific genes controlled by serum response factor (SRF). Myocardin is a cardiac and smooth musclespecific expressed transcriptional coactivator of SRF and is sufficient and necessary for smooth muscle gene expression. Here, we show that myocardin induces the acetylation of nucleosomal histones surrounding SRF-binding sites in the control regions of smooth muscle genes. The promyogenic activity of myocardin is enhanced by p300, a histone acetyltransferase that associates with the transcription activation domain of myocardin. Conversely, class II histone deacetylases interact with a domain of myocardin distinct from the p300-binding domain and suppress smooth muscle gene activation by myocardin. These findings point to myocardin as a nexus for positive and negative regulation of smooth muscle gene expression by changes in chromatin acetylation.

Abstract-In response to stress signals, postnatal cardiomyocytes undergo hypertrophic growth accompanied by activation of a fetal gene program, assembly of sarcomeres, and cellular enlargement. We show that hypertrophic signals stimulate the expression and transcriptional activity of myocardin, a cardiac and smooth muscle-specific coactivator of serum response factor (SRF). Consistent with a role for myocardin as a transducer of hypertrophic signals, forced expression of myocardin in cardiomyocytes is sufficient to substitute for hypertrophic signals and induce cardiomyocyte hypertrophy and the fetal cardiac gene program. Conversely, a dominant-negative mutant form of myocardin, which retains the ability to associate with SRF but is defective in transcriptional activation, blocks cardiomyocyte hypertrophy induced by hypertrophic agonists such as phenylephrine and leukemia inhibitory factor. Myocardin-dependent hypertrophy can also be partially repressed by histone deacetylase 5, a transcriptional repressor of myocardin. These findings identify myocardin as a nuclear effector of hypertrophic signaling pathways that couples stress signals to a transcriptional program for postnatal cardiac growth and remodeling. (Circ Res. 2006;98:1089-1097.)Key Words: cardiac hypertrophy Ⅲ cardiac myocytes Ⅲ cardiac transcription factors Ⅲ myocardin Ⅲ serum response factor Ⅲ transcription factors Ⅲ transcriptional regulation C ardiac myocytes proliferate rapidly during embryogenesis but lose their proliferative capacity soon after birth. 1 However, adult cardiac myocytes retain the ability to respond to mechanical, hemodynamic, hormonal, and pathologic stimuli by hypertrophic growth, defined by an increase in myocyte size or myofibrillar volume without a change in myocyte number. 2 Whereas cardiac hypertrophy allows the myocardium to adapt functional performance to alterations in workload associated with developmental maturation, physiological challenge, or injury, prolonged hypertrophy in response to stress signaling frequently progresses to heart failure with consequent sudden death attributable to cardiac arrythymias. 2 Cardiac hypertrophy is accompanied by the activation of a set of fetal cardiac genes that are normally expressed in the heart only before birth. 1,3 The reactivation of cardiac fetal genes in postnatal cardiomyocytes in response to hypertrophic signals suggests that the transcriptional program that controls cardiac gene expression during development may be redeployed to regulate hypertrophic cardiac growth. The MADS (MCM1, Agamous, Deficiens, SRF)-box transcription factor myocyte enhancer factor-2 (MEF2) and the zinc finger transcription factor GATA4 play important roles in cardiac development and in hypertrophic growth in response to stress, although the signaling pathways and underlying molecular mechanisms that modulate their activities are distinct. 4 -6 We have shown that MEF2 activity is stimulated by the signal-dependent dissociation from class II histone deacetylases (HDACs), which act as repressors...

SummaryThe cr88 mutant of Arabidopsis is a novel chlorate-resistant mutant that displays long hypocotyls in red light, but not in far red or blue light, and is delayed in the greening process. In cotyledons and young leaves, plastids are less developed compared with those of the wild type. In addition, a subset of light-regulated genes are under-expressed in this mutant. To understand the pleiotropic phenotypes of cr88, we isolated the CR88 gene through map-based cloning. We found that CR88 encodes a chloroplast-targeted 90-kDa heat shock protein (HSP90). The CR88 gene is expressed at highest levels during early post-germination stages and in leaves and reproductive organs. It is constitutively expressed but is also light and heat shock inducible. Chloroplast import experiments showed that the protein is localized to the stroma compartment of the chloroplast. The possible function of an HSP90 in the chloroplast and a plausible explanation of the pleiotropic phenotypes observed in cr88 are discussed.

Abstract-Transforming growth factor (TGF)-␤ 1 is an important cytokine involved in various diseases. However, the molecular mechanism whereby TGF-␤ 1 signaling modulates the regulatory network for smooth muscle gene transcription remains largely unknown. To address this question, we previously identified a Smad-binding element (SBE) in the SM22␣ promoter as one of the TGF-␤ 1 response elements. Here, we show that mutation of the SBE reduces the activation potential of a SM22␣ promoter in transgenic mice during embryogenesis. Chromatin immunoprecipitation assays reveal that TGF-␤ 1 induces Smad3 binding to the SM22␣ promoter in vivo. A multimerized SBE promoter responsive to TGF-␤ 1 signaling is highly activated by Smad3 but not by the closely related Smad2. Intriguingly, myocardin (Myocd), a known CArG box-dependent serum response factor coactivator, participates in Smad3-mediated TGF-␤ 1 signaling and synergistically stimulates Smad3-induced SBE promoter activity independent of the CArG box; no such synergy is seen with Smad2. Importantly, Myocd cooperates with Smad3 to activate the wild-type SM22␣, SM myosin heavy chain, and SM␣-actin promoters; they also activate the CArG box-mutated SM22␣ promoter as well as the CArG box-independent aortic carboxypeptidase-like protein promoter. Immunopreciptiation assays reveal that Myocd and Smad3 directly interact both in vitro and in vivo. Mutagenesis studies indicate that the C-terminal transactivation domains of Myocd and Smad3 are required for their functional synergy. These results reveal a novel regulatory mechanism whereby Myocd participates in TGF-␤ 1 signal pathway through direct interaction with Smad3, which binds to the SBEs. This is the first demonstration that Myocd can act as a transcriptional coactivator of the smooth muscle regulatory network in a CArG box-independent manner. (Circ Res. 2005;97:983-991.)Key Words: myocardin Ⅲ SM22␣ or transgelin Ⅲ Smad-binding site (SBE) Ⅲ Smad3 Ⅲ transforming growth factor-␤ 1 Ⅲ smooth muscle transcription

Cyclooxygenase-2 (COX-2) plays an important role in the inflammatory response induced by physiologic and stress stimuli. Exposure to diesel exhaust particulate matter (DEP) has been shown to induce pulmonary inflammation and exacerbate asthma and chronic obstructive pulmonary disease. DEP is a potent inducer of inflammatory reponses in human airway epithelial cells. The mechanism through which DEP inhalation induces inflammatory mediator expression is not understood. In this report, we demonstrate that DEP can induce the expression of COX-2 gene in a human bronchial epithelial cell line (BEAS-2B) at both transcriptional and protein levels. The induction of COX-2 gene expression involves chromatin modification, in particular acetylation and deacetylation of histones. We show that exposure to DEP increases the acetylation of histone H4 associated with the COX-2 promoter and causes degradation of histone deacetylase 1 (HDAC1). Further, we establish that HDAC1 plays a pivotal role in mediating the transcriptional activation of the COX-2 gene in BEAS-2B cells exposed to DEP, supported by evidence that the down-regulation of HDAC1 using siRNA leads to activation of COX-2 gene expression, whereas overexpression of HDAC1 results in its repression. Finally, DEP exposure induced recruitment of histone acetyltransferase (HAT) p300 to the promoter of the COX-2 gene, suggesting that acetylation is also important in regulating its expression in response to DEP exposure. These results show for the first time acetylation via selective degradation of HDAC1, and that recruitment of HAT plays an important role in DEP-induced expression of the COX-2 gene.

.-In vivo exposure to diesel exhaust particles (DEP) elicits acute inflammatory responses in the lung characterized by inflammatory cell influx and elevated expression of mediators such as cytokines and chemokines. Signal transducers and activators of transcription (STAT) proteins are a family of cytoplasmic transcription factors that are key transducers of signaling in response to cytokine and growth factor stimulation. One member of the STAT family, Stat3, has been implicated as a regulator of inflammation but has not been studied in regard to DEP exposure. The results of this study show that DEP induces Stat3 phosphorylation as early as 1 h following stimulation and that phosphorylated Stat3 translocates into the nucleus. Inhibition of epidermal growth factor receptor (EGFR) and Src activities by the inhibitors PD-153035 and PP2, respectively, abolished the activation of Stat3 by DEP, suggesting that Stat3 activation by DEP requires EGFR and Src kinase activation. The present study suggests that oxidative stress induced by DEP may play a critical role in activating EGFR signaling, as evidenced by the fact that pretreatment with antioxidant prevented the activation of EGFR and Stat3. These findings demonstrate that DEP inhalation can activate proinflammatory Stat3 signaling in vitro.signal transducer and activator of transcription 3; epidermal growth factor receptor; reactive oxygen species; human airway epithelial cells; diesel exhaust particles DIESEL EXHAUST PARTICULATES (DEP) emitted during the combustion of diesel fuel are a major contributor to airborne particulate matter (PM) mass in urban areas (7,37,50). DEP consist of a carbonaceous particle core onto which thousands of organic compounds can be adsorbed. DEP also contain notable quantities of noncarbonaceous inorganic species that are the result of small amount of fuel ash, ash components, and metallic additives present in lubrication oil and from engine wear. The organic compounds that are known to be proinflammatory or mutagenic include the polycyclic aromatic hydrocarbons (PAH), nitroaromatic compounds, quinones, aldehydes, heterocyclic compounds, and metal salts (10, 42). DEP emissions, which typically range from between 0.05 and 0.2 m in diameter, can persist in the air and are readily inhaled and deposited throughout the respiratory tract. Exposure to DEP has been associated with numerous adverse health outcomes, including pulmonary inflammation, increased susceptibility to respiratory infections, increased risk of cancer, and exacerbation of asthma and chronic obstructive pulmonary diseases (32). Although the mechanisms responsible for DEPinduced lung health effects remain to be fully elucidated, inflammatory responses are thought to play a pivotal role.Airway epithelial cells are known targets of PM inhalation and respond to DEP by producing numerous mediators involved in the lung immune and inflammatory responses, including production of cytokines, chemokines, and adhesion molecules (1, 4, 9, 51). The expression of such proinflammatory molec...

Myocardial differentiation is associated with the activation and expression of an array of cardiac specific genes. However, the transcriptional networks that control cardiac gene expression are not completely understood. Myocardin is a cardiac and smooth muscle-specific expressed transcriptional coactivator of Serum Response Factor (SRF) and is able to potently activate cardiac and smooth muscle gene expression during development. We hypothesize that myocardin discriminates between cardiac and smooth muscle specific genes by associating with distinct co-factors. Here, we show that myocardin directly interacts with Tbx5, a member of the T-box family of transcription factors involved in the Holt-Oram syndrome. Tbx5 synergizes with myocardin to activate expression of the cardiac specific genes atrial natriuretic factor (ANF) and alpha myosin heavy chain (α-MHC), but not that of smooth muscle specific genes SM22 or smooth muscle myosin heavy chain (SM-MHC). We found that this synergistic activation of shared target genes is dependent on the binding sites for Tbx5, T-box factor-Binding Elements (TBEs). Myocardin and Tbx5 physically interact and their interaction domains were mapped to the basic domain and the coil domain of myocardin and Tbx5, respectively. Our analysis demonstrates that the Tbx5G80R mutation, which leads to the Holt-Oram syndrome in humans, failed to synergize with myocardin to activate cardiac gene expression. These data uncover a key role for Tbx5 and myocardin in establishing the transcriptional foundation for cardiac gene activation and suggest that the interaction of myocardin and Tbx5 maybe involved in cardiac development and diseases.

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