Effect of a dominant negative ras on myocardial hypertrophy by using adenoviral-mediated gene transfer

Pracyk, J.B.; Hegland, Donald D.; Tanaka, Kōichi

doi:10.1016/s0039-6060(97)90033-7

Cited by 8 publications

(12 citation statements)

References 17 publications

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“…However, some stimuli, such as endothelin-1 and bacterial lipopolysaccharide, have been shown to activate MAPK in a Ras-independent manner (45,46). Our results indicate that hypoxia is similar to these stimuli, as expression of a dominant-negative Ras had no effect on the ability of EPAS1 to trans-activate the HRE-Luc reporter gene.…”

Section: Discussionmentioning

confidence: 64%

EPAS1 trans-Activation during Hypoxia Requires p42/p44 MAPK

Conrad

Freeman

Beitner‐Johnson

et al. 1999

Journal of Biological Chemistry

158

116

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Hypoxia is a common environmental stress that regulates gene expression and cell function. A number of hypoxia-regulated transcription factors have been identified and have been shown to play critical roles in mediating cellular responses to hypoxia. One of these is the endothelial PAS-domain protein 1 (EPAS1/HIF2-␣/HLF/ HRF). This protein is 48% homologous to hypoxia-inducible factor 1-␣ (HIF1-␣). To date, virtually nothing is known about the signaling pathways that lead to either EPAS1 or HIF1-␣ activation. Here we show that EPAS1 is phosphorylated when PC12 cells are exposed to hypoxia and that p42/p44 MAPK is a critical mediator of EPAS1 activation. Pretreatment of PC12 cells with the MEK inhibitor, PD98059, completely blocked hypoxiainduced trans-activation of a hypoxia response element (HRE) reporter gene by transfected EPAS1. Likewise, expression of a constitutively active MEK1 mimicked the effects of hypoxia on HRE reporter gene expression. However, pretreatment with PD98059 had no effect on EPAS1 phosphorylation during hypoxia, suggesting that MAPK targets other proteins that are critical for the trans-activation of EPAS1. We further show that hypoxia-induced trans-activation of EPAS1 is independent of Ras. Finally, pretreatment with calmodulin antagonists nearly completely blocked both the hypoxia-induced phosphorylation of MAPK and the EPAS1 trans-activation of HRE-Luc. These results demonstrate that the MAPK pathway is a critical mediator of EPAS1 activation and that activation of MAPK and EPAS1 occurs through a calmodulin-sensitive pathway and not through the GTPase, Ras. These results are the first to identify a specific signaling pathway involved in EPAS1 activation.Regulation of gene expression is a primary response by which cells adapt to changes in the environment. The mechanisms involved in regulation of gene expression in response to hypoxia are beginning to be understood. Transcription factors that are activated by hypoxia include the hypoxia-inducible factor (HIF1-␣), 1 c-fos, and CREB (1-4). HIF1-␣ has been shown to be critical for hypoxia-induced regulation of a number of genes, including glycolytic enzymes, vascular endothelial growth factor, and erythropoieitin (5-7). Recently, endothelial PAS-domain protein 1 (EPAS1, also known as HIF2-␣, HLF, and HRF) was identified as a hypoxia-inducible transcription factor (8 -10). EPAS1 is a basic helix-loop-helix transcription factor, which shares 48% sequence identity with HIF1-␣ (8). EPAS1 protein levels, like HIF1-␣ levels, are relatively low under basal conditions and accumulate upon exposure of cells to hypoxia (11). These factors then translocate to the nucleus and trans-activate target genes containing the sequence 5Ј-GCCCTACGTGCTGTCTCA-3Ј, which is commonly referred to as the hypoxia response element (HRE) (8, 12). EPAS1 is expressed in many tissues and is particularly abundant in the type I oxygen-sensing cells of the carotid body (13). Type I cells act as the primary O 2 sensors in mammals and are responsible for matching changes in art...

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Section: Discussionmentioning

confidence: 64%

EPAS1 trans-Activation during Hypoxia Requires p42/p44 MAPK

Conrad

Freeman

Beitner‐Johnson

et al. 1999

Journal of Biological Chemistry

158

116

View full text Add to dashboard Cite

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“…Cells-Ventricular neonatal myocytes were harvested from 2-3-dayold Sprague-Dawley rats by trypsin and collagenase digestion, purified by differential preplating, and cultured as described previously (21). Staining with MF-20 antibody to myosin heavy chain revealed the purity of the myocyte preparation to be in the range of 90 -95% (data not shown).…”

Section: Methodsmentioning

confidence: 99%

Expression of Id1 Results in Apoptosis of Cardiac Myocytes through a Redox-dependent Mechanism

Tanaka

Pracyk

Takeda

et al. 1998

Journal of Biological Chemistry

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We have constructed a recombinant adenovirus (Ad.Id1) that allows for efficient expression of the helixloop-helix protein Id1. After infection with Ad.Id1, neonatal cardiac myocytes display a significant reduction in viability, which was proportional to the level of Id1 expression. A similar effect was observed in adult myocytes. Morphological and biochemical assays demonstrated that Id1 expression resulted in myocyte apoptosis. In contrast, expression of Id1 in endothelial cells, vascular smooth muscle cells, or fibroblasts did not affect the viability of these cells. Along with the induction of apoptosis, the expression of Id1 in neonatal cardiac myocytes resulted in an increase in the level of intracellular reactive oxygen species. The source of these reactive oxygen species appears to be the mitochondria. Reducing the ambient oxygen concentration or treatment with a cell-permeant H 2 O 2 scavenger prevented Id1-stimulated apoptosis in cardiac myocytes. These results suggest that the expression of Id1 leads to the induction of apoptosis in cardiac myocytes through a redox-dependent mechanism.The Id family of proteins belongs to a class of nuclear proteins known as helix-loop-helix (HLH) 1 proteins, which regulate differentiation and tissue-specific gene expression. To date, four different Id family members have been identified (1-6). Although these four proteins share a high degree of homology, especially within the HLH domain, evidence suggests that they exert nonoverlapping functions (7-9).The HLH domain acts as a protein dimerization motif. Members of the HLH protein family which act to regulate transcription positively also contain an additional basic domain that mediates binding to DNA. In contrast, members of the Id family lack this domain and therefore are unable to bind DNA. These structural differences have led to a model in which Id proteins act in a dominant negative fashion to inhibit differentiation (1, 2). Under these circumstances, expression of high levels of Id1 would result in the binding and subsequent inactivation of basic domain HLH proteins.Consistent with this model of Id function is the observation that Id expression decreases when skeletal muscle cells differentiate (10). In addition, forced expression of Id has been shown to inhibit tissue-specific gene expression or differentiation of a variety of cell types (1, 10 -12). Previous reports have demonstrated that Id levels are high in embryonic hearts and decrease after birth (13-15). Such a shift in Id expression is consistent with its role as an inhibitor of differentiation. Although Id expression is low or absent in adult cardiac myocytes in culture, its expression can be reactivated by certain stimuli, such as ␣-adrenergic agonists (14, 15). A similar induction of Id expression has been demonstrated in skeletal muscle after treatment with the cardiotoxic chemotherapeutic agent doxorubicin (16,17).Although previous studies have demonstrated that forced expression of Id proteins can block differentiation, relatively little is known a...

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“…PKC-dependent ERK activation may occur in a Ras-independent manner in some cell types (Gutkind 1998;Yamaguchi et al 1995). Accordingly, a dominant negative mutant of Ras did not inhibit cardiac ERK activation by constitutively active G q (LaMorte et al 1994), endothelin-1 (Pracyk et al 1997;Yamazaki et al 1999) or angiotensin II but was reported to inhibit ERK activation via α 1 -adrenoceptors (Thorburn 1994). While inhibition of small G-proteins of the Rho family abolishes angiotensin II-induced pre-myofibril formation and partially inhibits mature myofibril formation (Aoki et al 1998), Rho inhibition does not block ERK activation by angiotensin II or α 1 -adrenoceptor agonists (Sah et al 1996).…”

Section: Activation Of Cardiac Erkmentioning

confidence: 99%