Expression of invasion genes encoded by the large 230-kb plasmid of ShigeUlajflxneri is controlled by the virB gene, which is itself activated by another regulator, virF. Transcription of the invasion genes is temperature regulated, since they are activated in bacteria grown at 37 but not at 30°C. Recently, we have shown that the thermoregulated expression of invasion genes is mediated by thermal activation of virB transcription (T. Tobe, S. Nagai, B. Adler, M. Yoshikawa, and C. Sasakawa, Mol. Microbiol. 5:887-893, 1991). It has also been shown that a mutation that inactivates H-NS, the product ofvirR (hns), derepresses transcription of virB. To elucidate the molecular mechanisms underlying virB activation, we determined the location of the transcription start site and found it to be 54 bp upstream of the 5' end of the virB coding sequence. Deletion analysis revealed that transcriptional activation by virF requires a DNA segment of 110 bp extending upstream of the transcription start site. By using a protein binding assay with crude extracts of S. flexneri harboring the malE'-'virF fusion gene, which was able to activate virB transcription, two protein species, one of 70 kDa (MaIE'-'VirF fusion) and another of 16 kDa (H-NS), were shown to bind specifically to the virB promoter region. DNA footprinting analysis indicated that the VirF fusion and H-NS proteins bound to the upstream sequence spanning from -17 to -117 and to the sequence from -20 to +20, in which virB transcription starts, respectively. In an in vitro transcription assay, the V`irF fusion protein was shown to activate virB transcription while the H-NS protein blocked it. virB activation was seen only when negatively supercoiled DNA was used as a template. In in vivo studies, virB transcription was significantly decreased by adding novobiocin, a gyrase inhibitor, into the culture medium while virB transcription was increased by mutating hns. These in vitro and in vivo studies indicated that transcription of virB is activated through VirF binding to the upstream sequence of the virB promoter in a DNA-topology-dependent manner and is directly repressed by H-NS binding to the virB transcription start site.

We have recently shown that mechanical stress induces cardiomyocyte hypertrophy partly through the enhanced secretion of angiotensin II (ATII). Endothelin-1 (ET-1) has been reported to be a potent growth factor for a variety of cells, including cardiomyocytes. In this study, we examined the role of ET-1 in mechanical stress-induced cardiac hypertrophy by using cultured cardiomyocytes of neonatal rats. ET-1 (10maximally induced the activation of both Raf-1 kinase and mitogen-activated protein (MAP) kinases at 4 and 8 min, respectively, followed by an increase in protein synthesis at 24 h. All of these hypertrophic responses were completely blocked by pretreatment with BQ123, an antagonist selective for the ET-1 type A receptor subtype, but not by BQ788, an ET-1 type B receptorspecific antagonist. BQ123 also suppressed stretch-induced activation of MAP kinases and an increase in phenylalanine uptake by approximately 60 and 50%, respectively, but BQ788 did not. ET-1 was constitutively secreted from cultured cardiomyocytes, and a significant increase in ET-1 concentration was observed in the culture medium of cardiomyocytes after stretching for 10 min. After 24 h, an ϳ3-fold increase in ET-1 concentration was observed in the conditioned medium of stretched cardiomyocytes compared with that of unstretched cardiomyocytes. ET-1 mRNA levels were also increased at 30 min after stretching. Moreover, ET-1 and ATII synergistically activated Raf-1 kinase and MAP kinases in cultured cardiomyocytes. In conclusion, mechanical stretching stimulates secretion and production of ET-1 in cultured cardiomyocytes, and vasoconstrictive peptides such as ATII and ET-1 may play an important role in mechanical stress-induced cardiac hypertrophy.Cardiac hypertrophy, a major underlying cause of heart diseases such as myocardial infarction and cardiac arrhythmias (1), is formed when increased external stimuli such as hemodynamic overload and neurohumoral factors are continuously imposed on cardiac myocytes (2, 3). These external stimuli are generally transduced into the nucleus through protein kinase cascades of phosphorylation (4), and Raf-1 kinase (Raf-1) 1 (5) and mitogen-activated protein (MAP) kinases (6, 7) are important components in these cascades. We have recently reported that stretching of cardiomyocytes sequentially activates Raf-1 and MAP kinases, followed by an increase in protein synthesis (8). Interestingly, all of these events were partially suppressed by a specific antagonist of the angiotensin II (ATII) type 1 receptor, CV11974 (9). These results suggest that mechanical stress exemplified by stretching might stimulate the secretion of ATII from cardiomyocytes and that ATII participates in the activation of the protein kinase cascades and the production of cardiomyocyte hypertrophy through the ATII type 1 receptor. However, because the inhibition of these hypertrophic events by CV11974 is incomplete, factors other than ATII should also be involved in cardiomyocyte hypertrophy induced by mechanical stress.Endothelin-1 (ET-1) ...

We have previously shown that mechanical stress induces activation of protein kinases and increases in specific gene expression and protein synthesis in cardiac myocytes, all of which are similar to those evoked by humoral factors such as growth factors and hormones. Many lines of evidence have suggested that angiotensin II (Ang II) plays a vital role in cardiac hypertrophy, and it has been reported that secretion of Ang II from cultured cardiac myocytes was induced by mechanical stretch. To examine the role of Ang II in mechanical stress-induced cardiac hypertrophy, we stretched neonatal rat cardiac myocytes in the absence or presence of the Ang II receptor antagonists saralasin (an antagonist of both type 1 and type 2 receptors), CV-11974 (a type 1 receptor-specific antagonist), and PD123319 (a type 2 receptor-specific antagonist). Stretching cardiac myocytes by 20% using deformable silicone dishes rapidly increased the activities of mitogen-activated protein (MAP) kinase kinase activators and MAP kinases. Both saralasin and CV-11974 partially inhibited the stretch-induced increases in the activities of both kinases, whereas PD123319 showed no inhibitory effects. Stretching cardiac myocytes increased amino acid incorporation, which was also inhibited by approximately 70% with the pretreatment by saralasin or CV-11974. When the culture medium conditioned by stretching cardiocytes was transferred to nonstretched cardiac myocytes, the increase in MAP kinase activity was observed, and this increase was completely suppressed by saralasin or CV-11974. These results suggest that Ang II plays an important role in mechanical stress-induced cardiac hypertrophy and that there are also other (possibly nonsecretory) factors to induce hypertrophic responses.

Background-Many studies have suggested that the renin-angiotensin system plays an important role in the development of pressure overload-induced cardiac hypertrophy. Moreover, it has been reported that pressure overload-induced cardiac hypertrophy is completely prevented by ACE inhibitors in vivo and that the stored angiotensin II (Ang II) is released from cardiac myocytes in response to mechanical stretch and induces cardiomyocyte hypertrophy through the Ang II type 1 receptor (AT 1 ) in vitro. These results suggest that the AT 1 -mediated signaling is critical for the development of mechanical stress-induced cardiac hypertrophy. Methods and Results-To determine whether AT 1 -mediated signaling is indispensable for the development of pressure overload-induced cardiac hypertrophy, pressure overload was produced by constricting the abdominal aorta of AT 1A knockout (KO) mice. Quantitative reverse transcriptase-polymerase chain reaction revealed that the cardiac AT 1 (probably AT 1B ) mRNA levels in AT 1A KO mice were Ͻ10% of those of wild-type (WT) mice and were not affected by pressure overload. Chronic treatment with subpressor doses of Ang II increased left ventricular mass in WT mice but not in KO mice. Pressure overload, however, fully induced cardiac hypertrophy in KO as well as WT mice. There were no significant differences between WT and KO mice in expression levels of fetal-type cardiac genes, in the left ventricular wall thickness and systolic function as revealed by the transthoracic echocardiogram, or in the histological changes such as myocyte hypertrophy and fibrosis.

The Rho family GTP-binding proteins play a critical role in a variety of cytoskeleton-dependent cell functions. In this study, we examined the role of Rho family G proteins in muscle differentiation. Dominant negative forms of Rho family proteins and RhoGDI, a GDP dissociation inhibitor, suppressed transcription of muscle-specific genes, while mutationally activated forms of Rho family proteins strongly activated their transcription. C2C12 cells overexpressing RhoGDI (C2C12RhoGDI cells) did not differentiate into myotubes, and expression levels of myogenin, MRF4, and contractile protein genes but not MyoD and myf5 genes were markedly reduced in C2C12RhoGDI cells. The promoter activity of the myogenin gene was suppressed by dominant negative mutants of Rho family proteins and was reduced in C2C12RhoGDI cells. Expression of myocyte enhancer binding factor 2 (MEF2), which has been reported to be required for the expression of the myogenin gene, was reduced at the mRNA and protein levels in C2C12RhoGDI cells. These results suggest that the Rho family proteins play a critical role in muscle differentiation, possibly by regulating the expression of the myogenin and MEF2 genes.Myogenic basic helix-loop-helix (bHLH) proteins are master regulatory proteins that activate the transcription of many muscle-specific genes during myogenesis (reviewed in references 60 and 89). Each of the four myogenic bHLH proteins, MyoD (17), myogenin (18, 90), myf5 (7), and MRF4 (8, 47, 67), can activate the skeletal myogenic program when introduced into a variety of cells derived from all three germ layers of the embryo. The bHLH motif mediates dimerization of myogenic factors with ubiquitous bHLH proteins such as E12/E47, and these heterodimeric complexes bind to a conserved DNA sequence known as the E box, which is present in the promoters and enhancers of most muscle-specific genes (54). Myocyte enhancer binding factor 2 (MEF2), which is a member of the MADS box family, also plays an important role in muscle differentiation (reviewed in reference 61). MEF2 activates transcription by binding to the consensus sequence, called the MEF2-binding site, which is also found in the control regions of numerous muscle-specific genes (23, 64). In embryos with loss-of-function mutations of the single mef2 gene in Drosophila (D-mef2), somatic, cardiac, and visceral muscle cells did not differentiate (6,38,66). These results indicated that MEF2 is necessary for the differentiation of all types of muscle cells. MEF2 and myogenic bHLH proteins have been suggested to activate mutual expression in an autoregulatory network and maintain the expression of muscle-specific genes (5,16,31,43,56). Moreover, a recent study demonstrated that MEF2 and myogenic bHLH proteins synergistically activate expressions of muscle-specific genes via protein-protein interactions between DNA-binding domains of these heterologous classes of transcription factors (51). In addition, it has been reported that a variety of factors, such as fibroblast growth factor, transforming grow...