scholarly journals The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element

2000 ◽  
Vol 278 (6) ◽  
pp. H1769-H1774 ◽  
Author(s):  
Michael R. Morissette ◽  
Valerie P. Sah ◽  
Christopher C. Glembotski ◽  
Joan Heller Brown
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Common Element ◽  
Luciferase Reporter ◽  
Serum Response Element ◽  
Transcriptional Responses ◽  
Response Element ◽  
Serum Response

The low-molecular-weight GTP-binding protein RhoA mediates hypertrophic growth and atrial natriuretic factor (ANF) gene expression in neonatal rat ventricular myocytes. Neither the effector nor the promoter elements through which Rho exerts its regulatory effects on ANF gene expression have been elucidated. When constitutively activated forms of Rho kinase and two protein kinase C-related kinases, PKN (PRK1) and PRK2, were compared, only PKN generated a robust stimulation of a luciferase reporter gene driven by a 638-bp fragment on the ANF promoter. This ANF promoter fragment contains a proximal serum response element (SRE) and an Sp-1-like element required for the transcriptional response to phenylephrine (PE). This response was inhibited by dominant negative Rho. The ability of dominant negative Rho to inhibit the response to PE and the ability of PKN to stimulate ANF reporter gene expression were both lost when the SRE was mutated. Mutation of the Sp-1-like element also attenuated the response to PKN. A minimal promoter driven by ANF SRE sequences was sufficient to confer Rho- and PKN-mediated gene expression. Interestingly, PKN preferentially stimulated the ANF versus the c- fos SRE reporter gene. Thus PKN and Rho are able to regulate transcriptional activation of the ANF SRE by a common element that could implicate PKN as a downstream effector of Rho in transcriptional responses associated with hypertrophy.

scholarly journals Regulation of GLUT4 gene expression by SREBP-1c in adipocytes

2006 ◽  
Vol 399 (1) ◽  
pp. 131-139 ◽  
Author(s):  
Seung-Soon Im ◽  
Sool-Ki Kwon ◽  
Seung-Youn Kang ◽  
Tae-Hyun Kim ◽  
Ha-Il Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Glucose Transporter ◽  
Regulatory Element ◽  
Diabetic Rats ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Response Element ◽  
Glut4 Gene Expression

Expression of the GLUT4 (glucose transporter type 4 isoform) gene in adipocytes is subject to hormonal or metabolic control. In the present study, we have characterized an adipose tissue transcription factor that is influenced by fasting/refeeding regimens and insulin. Northern blotting showed that refeeding increased GLUT4 mRNA levels for 24 h in adipose tissue. Consistent with an increased GLUT4 gene expression, the mRNA levels of SREBP (sterol-regulatory-element-binding protein)-1c in adipose tissue were also increased by refeeding. In streptozotocin-induced diabetic rats, insulin treatment increased the mRNA levels of GLUT4 in adipose tissue. Serial deletion, luciferase reporter assays and electrophoretic mobility-shift assay studies indicated that the putative sterol response element is located in the region between bases −109 and −100 of the human GLUT4 promoter. Transduction of the SREBP-1c dominant negative form to differentiated 3T3-L1 adipocytes caused a reduction in the mRNA levels of GLUT4, suggesting that SREBP-1c mediates the transcription of GLUT4. In vivo chromatin immunoprecipitation revealed that refeeding increased the binding of SREBP-1 to the putative sterol-response element in the GLUT4. Furthermore, treating streptozotocin-induced diabetic rats with insulin restored SREBP-1 binding. In addition, we have identified an Sp1 binding site adjacent to the functional sterol-response element in the GLUT4 promoter. The Sp1 site appears to play an additive role in SREBP-1c mediated GLUT4 gene upregulation. These results suggest that upregulation of GLUT4 gene transcription might be directly mediated by SREBP-1c in adipose tissue.

scholarly journals Stimulation of gene expression in neonatal rat ventricular myocytes by Ras is mediated by Ral guanine nucleotide dissociation stimulator (Ral.GDS) and phosphatidylinositol 3-kinase in addition to Raf

1998 ◽  
Vol 335 (2) ◽  
pp. 241-246 ◽  
Author(s):  
Stephen J. FULLER ◽  
Stephen G. FINN ◽  
Julian DOWNWARD ◽  
Peter H. SUGDEN
Keyword(s):  
Cell Size ◽  
Reporter Gene ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Guanine Nucleotide ◽  
Phosphatidylinositol 3 Kinase ◽  
Transcriptional Responses ◽  
Response Element ◽  
Hypertrophic Response ◽  
Phosphatidylinositol 3

Treatment of cultured neonatal ventricular myocytes with oncogenic Ras increases their size and stimulates the re-expression of genes which are normally restricted to the fetal stage of ventricular development, including atrial natriuretic factor (ANF) and skeletal muscle (SkM)-α-actin. To determine which signalling pathways mediate these responses, myocytes were transfected with oncogenic (V12) Ras mutants which interact selectively with different effectors and their effects on luciferase (LUX) reporter plasmids were examined. V12 human Ras (V12HRas), itself, activated ANF–LUX 9.6-fold, whereas mutants of V12HRas, which selectively stimulate Ral guanine nucleotide dissociation stimulator (Ral.GDS) (E37G), c-Raf (D38E) and phosphatidylinositol 3-kinase (PI-3-K; Y40C) enhanced ANF–LUX expression 3.0-, 3.7- and 1.7-fold respectively. The full response of ANF–LUX to V12HRas was restored by using a combination of the individual effector domain mutants. Likewise, SkM-α-actin–LUX expression was activated 12.0-, 3.5-, 4.5- and 3.0-fold by V12HRas, E37G, D38E and Y40C respectively, and a similar pattern of activation was also observed using a c-fosserum-response element–LUX reporter gene. Cell size was also increased by each of the mutants, but simultaneous expression of all three mutant constructs was needed to reconstitute the full effect of V12HRas on cell size (50% increase). Transfection with a constitutively active mutant of PI-3-K (p110K227E) stimulated ANF–LUX, SkM-α-actin–LUX, c-fos-serum-response element–LUX and Rous sarcoma virus–LUX by 3.1-, 3.2-, 2.1- and 2.9-fold respectively, but the co-transfected cytomegalovirus-β-galactosidase reporter gene was activated to a similar extent (1.9-fold). These results suggest that Raf, Ral.GDS and PI-3-K can all transduce transcriptional responses to V12HRas, but that the specific induction of genes associated with the hypertrophic response is not mediated through PI-3-K.

scholarly journals CRE and SRE mediate LPA-induced CCN1 transcription in mouse aortic smooth muscle cells

2017 ◽  
Vol 95 (3) ◽  
pp. 275-280
Author(s):  
Quanlin Dou ◽  
Feng Hao ◽  
Longsheng Sun ◽  
Xuemin Xu ◽  
Mei-Zhen Cui
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Reporter Gene ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Response Element ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Serum Response

Lysophosphatidic acid (LPA), one component of oxidized low-density lipoprotein (ox-LDL), is a potent bioactive phospholipid. Our recent data reveal that LPA induces matricellular protein CCN1 (also known as Cyr61) expression in aortic smooth muscle cells (SMCs) and that CCN1 bridges LPA and integrin signaling pathways leading to SMC migration. Whether and how LPA regulates the transcriptional machinery of the CCN1 gene are unknown. In this study, we found that LPA markedly induces CCN1 mRNA expression in SMCs. Using deleting mutation and reporter gene strategies, we demonstrated regions from –2038 to –1787 and from –101 to +63 of the CCN1 promoter contain the essential regulatory elements. The serum response element (SRE) and cyclic AMP-response element (CRE) are located in these regions. LPA induced time-dependent phosphorylation of serum response factor (SRF) and CRE-binding protein (CREB) in mouse SMCs. Luciferase assays of a series of deleted, mutated CCN1 promoter-reporter gene constructs and dominant negative construct revealed the distal SRE and the proximal CRE in the CCN1 promoter are required for LPA-induced CCN1 gene expression. Our results imply that elevated LPA levels may trigger SMC migration and exacerbate restenosis and atherosclerotic lesions through the induced CCN1, which communicates with a set of plasma membrane proteins and intracellular kinases.

scholarly journals Voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases propagate signals from endothelin-1 receptors to the c-fos promoter.

1996 ◽  
Vol 16 (10) ◽  
pp. 5915-5923 ◽  
Author(s):  
Y Wang ◽  
M S Simonson
Keyword(s):  
Gene Expression ◽  
Protein Kinases ◽  
Ectopic Expression ◽  
Promoter Activation ◽  
Serum Response Element ◽  
Response Element ◽  
Endothelin 1 ◽  
Dependent Protein ◽  
Serum Response ◽  
Ca2 Channels

Endothelin-1 (ET-1) triggers poorly understood nuclear signaling cascades that control gene expression, cell growth, and differentiation. To better understand how ET-1 regulates gene expression, we asked whether voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases (CaMKs) propagate signals from ET-1 receptors to the c-fos promoter in mesangial cells. Ca2+ influx through voltage-insensitive Ca2+ channels, one of the earliest postreceptor events in ET-1 signaling, mediated induction of c-fos mRNA and activation of the c-fos promoter by ET-1. A CaMK inhibitor (KN-93) blocked activation of the c-fos promoter by ET-1. Ectopic expression of CaMKII potentiated stimulation by ET-1, providing further evidence that CaMKs contribute to c-fos promoter activation by ET-1. The c-fos serum response element was necessary but not sufficient for CaMKII to activate the c-fos promoter. Activation of the c-fos promoter by ET-1 and CaMKII also required the FAP cis element, an AP-1-like sequence adjacent to the serum response element. Thus, voltage-insensitive Ca2+ channels and CaMKs apparently propagate ET-1 signals to the c-fos promoter that require multiple, interdependent cis elements. Moreover, these experiments suggest an important role for voltage-insensitive Ca2+ channels in nuclear signal transduction in nonexcitable cells.

scholarly journals Estradiol Reduces Nonclassical Transcription at Cyclic Adenosine 3′,5′-Monophosphate Response Elements in Glioma Cells Expressing Estrogen Receptor Alpha

Endocrinology ◽  
2006 ◽  
Vol 147 (4) ◽  
pp. 1796-1804 ◽  
Author(s):  
Andrew J. Mhyre ◽  
Robert A. Shapiro ◽  
Daniel M. Dorsa
Keyword(s):  
Gene Expression ◽  
Estrogen Receptor ◽  
Reporter Gene ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Estradiol Treatment ◽  
Camp Response Element ◽  
Response Element ◽  
Functional Changes ◽  
Rapid Signaling

Estradiol can protect the brain from a variety of insults by activating membrane-initiated signaling pathways, and thereby modulate gene expression and lead to functional changes in neurons. These direct neuronal effects of the hormone have been well documented; however, it is less understood what effects estradiol may have on nonneuronal cells of the central nervous system. There is evidence that estradiol levels can induce the release of glial-derived growth factors and other cytokines, suggesting that estradiol may both directly and indirectly protect neurons. To determine whether 17β-estradiol (E2) can activate rapid signaling and modulate nonclassical transcription in astrocytes, we stably transfected the C6 rat glioblastoma cell line with human estrogen receptor (ER) α (C6ERα) or rat ERβ (C6ERβ). Introduction of a cAMP response element-luciferase reporter gene into C6, C6ERα, and C6ERβ cells leads to the observation that E2 treatment reduced isoproterenol-stimulated luciferase activity by 35% in C6ERα but had no effect on reporter gene expression in C6ERβ or untransfected C6 cells. A similar effect was seen with a membrane-impermeable estrogen (E2-BSA), suggesting the modulation of nonclassical transcription by estradiol treatment is mediated by the activation of a membrane-initiated signaling pathway. Furthermore, pretreatment with wortmannin (phosphatidylinsositol 3-kinase) or U73122 (phospholipase C) attenuated the E2-induced reduction in nonclassical transcription. We conclude that E2 treatment reduces cAMP response element-mediated transcription in glioma cells expressing ERα and that this reduction is dependent on the activation of membrane-initiated signaling. These findings suggest a novel model of estrogen rapid signaling in astrocytes that leads to modulation of nonclassical transcription.

scholarly journals Cas Mediates Transcriptional Activation of the Serum Response Element by Src

1999 ◽  
Vol 19 (10) ◽  
pp. 6953-6962 ◽  
Author(s):  
Yaron Hakak ◽  
G. Steven Martin
Keyword(s):  
Transcriptional Activation ◽  
Tyrosine Phosphatase ◽  
Sh3 Domain ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Adapter Protein ◽  
Binding Region ◽  
Serum Response Element ◽  
Response Element ◽  
Serum Response

ABSTRACT The Src substrate p130Cas is a docking protein containing an SH3 domain, a substrate domain that contains multiple consensus SH2 binding sites, and a Src binding region. We have examined the possibility that Cas plays a role in the transcriptional activation of immediate early genes (IEGs) by v-Src. Transcriptional activation of IEGs by v-Src occurs through distinct transcriptional control elements such as the serum response element (SRE). An SRE transcriptional reporter was used to study the ability of Cas to mediate Src-induced SRE activation. Coexpression of v-Src and Cas led to a threefold increase in SRE-dependent transcription over the level induced by v-Src alone. Cas-dependent activation of the SRE was dependent on the kinase activity of v-Src and the Src binding region of Cas. Signaling to the SRE is promoted by a serine-rich region within Cas and inhibited by the Cas SH3 domain. Cas-dependent SRE activation was accompanied by an increase in the level of active Ras and in the activity of the mitogen-activated protein kinase (MAPK) Erk2; these changes were blocked by coexpression of dominant-negative mutants of the adapter protein Grb2. SRE activation was abrogated by coexpression of dominant-negative mutants of Ras, MAPK kinase (Mek1), and Grb2. Coexpression of Cas with v-Src enhanced the association of Grb2 with the adapter protein Shc and the protein tyrosine phosphatase Shp-2; coexpression of Shc or Shp-2 mutants significantly reduced SRE activation by Cas and v-Src. Cas-induced Grb2 association with Shp-2 and Shc may account for the Cas-dependent activation of the Ras/Mek/Erk pathway and SRE-dependent transcription. 14-3-3 proteins may also play a role in Cas-mediated signaling to the SRE. Overexpression of Cas was found to modestly enhance epidermal growth factor (EGF)-induced activation of the SRE. A Cas mutant lacking the Src binding region did not potentiate the EGF response, suggesting that Cas enhances EGF signaling by binding to endogenous cellular Src or another Src family member. These observations implicate Cas as a mediator of Src-induced transcriptional activation.

scholarly journals A Low-Affinity Serum Response Element Allows Other Transcription Factors To Activate Inducible Gene Expression in Cardiac Myocytes

1999 ◽  
Vol 19 (3) ◽  
pp. 1841-1852 ◽  
Author(s):  
Wirt A. Hines ◽  
Jacqueline Thorburn ◽  
Andrew Thorburn
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Consensus Sequence ◽  
Serum Response Element ◽  
Cardiac Muscle Cells ◽  
Response Element ◽  
Inducible Gene Expression ◽  
Hypertrophic Growth ◽  
Serum Response ◽  
Inducible Gene

ABSTRACT Hypertrophic growth of cardiac muscle cells is induced by a variety of physiological and pathological stimuli and is associated with a number of changes, including activation of genes such as atrial natriuretic factor. We found that two serum response element (SRE)-like DNA elements, one of which does not meet the consensus sequence and binds serum response factor (SRF) with low affinity, regulate the activity of this promoter. Surprisingly, the ability to induce the promoter by two different physiologic stimuli, as well as various activated transcription factors, including SRF-VP16, was primarily dependent upon the nonconsensus rather than the consensus SRE. This SRE controls the induction of gene expression via an unusual mechanism in that it is required to allow some, but not all, active transcription factors at unrelated sites on the promoter to stimulate gene expression. Thus, in addition to regulation of SRF activity by growth stimuli, regulation of a low-affinity SRE element controls inducible gene expression by modulating the ability of other transcription factors to stimulate the transcription machinery.

scholarly journals Loss of serum response element-binding activity and hyperphosphorylation of serum response factor during cellular aging.

1994 ◽  
Vol 14 (7) ◽  
pp. 4991-4999 ◽  
Author(s):  
P W Atadja ◽  
K F Stringer ◽  
K T Riabowol
Keyword(s):  
Gene Expression ◽  
Serum Response Factor ◽  
Cellular Aging ◽  
Response Factor ◽  
Serum Response Element ◽  
Early Passage ◽  
Core Element ◽  
Response Element ◽  
Serum Response

Human diploid fibroblasts undergo a limited number of population doublings in vitro and are used widely as a model of cellular aging. Despite growing evidence that cellular aging occurs as a consequence of altered gene expression, little is known about the activity of transcription factors in aging cells. Here, we report a dramatic reduction in the ability of proteins extracted from the nuclei of near-senescent fibroblasts to bind the serum response element which is necessary for serum-induced transcription of the c-fos gene. In contrast, the activities of proteins binding to the RNA polymerase core element, TATA, as well as to the cyclic AMP response element were maintained during cellular aging. While no major differences in the expression of the serum response factor (SRF) that binds the serum response element were seen between early-passage and late-passage cells, hyperphosphorylation of SRF was observed in near-senescent cells. Furthermore, removal of phosphatase inhibitors during the isolation of endogenous nuclear proteins restored the ability of SRF isolated from old cells to bind the SRE. These data, therefore, indicate that hyperphosphorylation of SRF plays a role in altering the ability of this protein to bind to DNA and regulate gene expression in senescent cells.

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