A variety of stresses on the heart initiate a number of subcellular signaling pathways, which finally reach the nuclei of cardiac myocytes and cause myocyte hypertrophy with heart failure. However, common nuclear pathways that lead to this state are unknown. A zinc finger protein, GATA-4, is one of the transcription factors that mediate changes in gene expression during myocardial-cell hypertrophy. p300 not only acts as a transcriptional coactivator of GATA-4, but also possesses an intrinsic histone acetyltransferase activity. In primary cardiac myocytes derived from neonatal rats, we show that stimulation with phenylephrine increased an acetylated form of GATA-4 and its DNA-binding activity, as well as expression of p300. A dominant-negative mutant of p300 suppressed phenylephrine-induced nuclear acetylation, activation of GATA-4-dependent endothelin-1 promoters, and hypertrophic responses, such as increase in cell size and sarcomere organization. In sharp contrast to the activation of cardiac MEK-1, which phosphorylates GATA-4 and causes compensated hypertrophy in vivo, p300-mediated acetylation of mouse cardiac nuclear proteins, including GATA-4, results in marked eccentric dilatation and systolic dysfunction. These findings suggest that p300-mediated nuclear acetylation plays a critical role in the development of myocyte hypertrophy and represents a pathway that leads to decompensated heart failure.Heart failure arises from a number of diverse primary cardiovascular disorders and is associated with significant morbidity and mortality. Therefore, elucidating the mechanisms of this disease is of clinical importance. Previous studies have demonstrated that a variety of stresses on the heart activate neuronal and hormonal factors, such as the renin-angiotensin system and factors regulating the sympathetic nervous systems. These factors initiate a number of subcellular signaling pathways, which finally reach the nuclei of cardiac myocytes and change the pattern of gene expression associated with hypertrophy (reviewed in references 14 and 53). In order to establish appropriate therapy for heart failure, it is critical to identify a common nuclear pathway which can be targeted by pharmacological agents in the future.We have been interested in transcription factors that mediate changes in gene expression during myocardial-cell hypertrophy. A zinc finger protein, GATA-4, is one such factor and is required for transcriptional activation of cardiac genes whose expression is upregulated during myocardial-cell hypertrophy (22, 25; reviewed in reference 46). While overexpression of GATA-4 in cardiac myocytes causes hypertrophy, expression of a dominant-negative form of GATA-4 inhibits Gq protein-coupled receptor agonist-induced hypertrophy (37). During myocardial-cell hypertrophy, GATA-4 is phosphorylated at a serine residue and shows increased DNA-binding ability (38,47). Phosphorylation of cardiac GATA-4 requires activation of MEK1/extracellular signal-regulated kinase (ERK) 1/2. On the other hand, activation of MEK1 in...

Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat–containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5–deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171–175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168–171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.

Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-β binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-β because TGF-β-free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-β-bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.connective tissue | development | extracellular matrix

Great arteries, as well as lungs and skin, contain elastic fibers as important components to maintain their physiological functions. Although recent studies have revealed that a glycoprotein fibulin-4 (FBLN4) is indispensable for the assembly of mature elastic fibers, it remains to be elucidated how FBLN4 takes part in elastogenesis. Here, we report a dose-dependent requirement for FBLN4 in the development of the elastic fibers in arteries, and a specific role of FBLN4 in recruiting the elastin-cross-linking enzyme, lysyl oxidase (LOX). Reduced expression of Fbln4, which was achieved with a smooth muscle-specific Cre-mediated gene deletion, caused arterial stiffness. Electron-microscopic examination revealed disorganized thick elastic laminae with aberrant deposition of elastin. Aneurysmal dilation of the ascending aorta was found when the Fbln4 expression level was reduced to an even lower level, whereas systemic Fbln4 null mice died perinatally from rupture of the diaphragm. We also found a specific interaction between FBLN4 and the propeptide of LOX, which efficiently promotes assembly of LOX onto tropoelastin. These data suggest a mechanism of elastogenesis, in which a sufficient amount of FBLN4 is essential for tethering LOX to tropoelastin to facilitate cross-linking.development ͉ elastin ͉ extracellular matrix

Differentiation of embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) govern gene expression patterns by being recruited to target genes through association with specific transcription factors. One of the HATs, p300, serves as a coactivator of cardiac-specific transcription factors such as GATA-4. The HAT activity of p300 is required for acetylation and DNA binding of GATA-4 and its full transcriptional activity as well as for promotion of a transcriptionally active chromatin configuration. However,

Elastic fibers play the principal roles in providing elasticity and integrity to various types of human organs, such as the arteries, lung, and skin. However, the molecular mechanism of elastic fiber assembly that leads to deposition and crosslinking of elastin along microfibrils remains largely unknown. We have previously shown that developing arteries and neural crest EGF-like protein (DANCE) (also designated fibulin-5) is essential for elastogenesis by studying DANCE-deficient mice. Here, we report the identification of latent transforming growth factor-b-binding protein 2 (LTBP-2), an elastic fiber-associating protein whose function in elastogenesis is not clear, as a DANCEbinding protein. Elastogenesis assays using human skin fibroblasts reveal that fibrillar deposition of DANCE and elastin is largely dependent on fibrillin-1 microfibrils. However, downregulation of LTBP-2 induces fibrillin-1-independent fibrillar deposition of DANCE and elastin. Moreover, recombinant LTBP-2 promotes deposition of DANCE onto fibrillin-1 microfibrils. These results suggest a novel regulatory mechanism of elastic fiber assembly in which LTBP-2 regulates targeting of DANCE on suitable microfibrils to form elastic fibers.

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter ؊1 ) rather than GGOH (0.2 mg liter ؊1 ) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter ؊1 GGOH and 6.5 mg liter ؊1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter ؊1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.(E,E,E)-Geranylgeraniol (GGOH) can be used as an important ingredient for perfumes and as a desirable raw material for synthesizing vitamins A and E (4, 13). It is also known to induce apoptosis in various cancer and tumor cell lines (24,36). GGOH is the dephosphorylated derivative of (E,E,E)-geranylgeranyl diphosphate (GGPP) (Fig. 1). GGPP is a significant intermediate of ubiquinone and carotenoid biosyntheses, especially in carotenoid-producing microorganisms and plant cells. It is also utilized as the lipid anchor of geranylgeranylated proteins. In the yeast Saccharomyces cerevisiae, GGPP is synthesized by GGPP synthase (GGPS), encoded by the BTS1 gene, which catalyzes the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) rather than the successive addition of IPP molecules to dimethylallyl diphosphate, geranyl diphosphate, and FPP that is detected in mammalian tissues (14). Biologically synthesized GGOH comprises only (E,E,E)-geometric isomers, and only the (E,E,E)-isomers have significant biological activities (23). The chemically synthesized form is usually obtained as mixtures of (E)-and (Z)-isomers and thus has lower potency. Therefore, there is a greater possibility of attaining efficient production of (E,E,E)-GGOH through fermentative production.Some yeast strains accumulate ergosterol up to 4.6% d...

Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase–mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation.