scholarly journals Regulation of Cyr61 gene expression by mechanical stretch through multiple signaling pathways

2001 ◽  
Vol 281 (5) ◽  
pp. C1524-C1532 ◽  
Author(s):  
Isao Tamura ◽  
Joel Rosenbloom ◽  
Edward Macarak ◽  
Brahim Chaqour
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Rho Kinase ◽  
Mechanical Stretch ◽  
Selective Inhibition ◽  
Early Gene ◽  
Actin Dynamics ◽  
Mrna Levels ◽  
Bladder Smooth Muscle ◽  
Pharmacological Agents

The cysteine-rich protein 61 (Cyr61) is a signaling molecule with functions in cell migration, adhesion, and proliferation. This protein is encoded by an immediate early gene whose expression is mainly induced by serum growth factors. Here we show that Cyr61 mRNA levels increase sharply in response to cyclic mechanical stretch applied to cultured bladder smooth muscle cells. Stretch-induced changes of Cyr61 transcripts were transient and accompanied by an increase of the encoded protein that localized mainly to the cytoplasm and nucleus of the cells. With the use of pharmacological agents that interfere with known signaling pathways, we show that transduction mechanisms involving protein kinase C and phosphatidylinositol 3-kinase activation partly blocked stretch-induced Cyr61 gene expression. Selective inhibition of Rho kinase pathways altered this stretch effect as well. Meanwhile, using inhibitors of the actin cytoskeleton, we show that Cyr61 gene expression is sensitive to mechanisms that sense actin dynamics. These results establish the regulation of Cyr61 gene by mechanical stretch and provide clues to the key signaling molecules involved in this process.

Cyr61 mediates the expression of VEGF, αv-integrin, and α-actin genes through cytoskeletally based mechanotransduction mechanisms in bladder smooth muscle cells

2005 ◽  
Vol 98 (6) ◽  
pp. 2344-2354 ◽  
Author(s):  
Dongming Zhou ◽  
David J. Herrick ◽  
Joel Rosenbloom ◽  
Brahim Chaqour
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Signaling Pathways ◽  
Target Genes ◽  
Contractile Function ◽  
Mechanical Strain ◽  
Cyclic Stretch ◽  
Actin Dynamics ◽  
Bladder Smooth Muscle ◽  
Static Conditions

Application of cyclic strain to bladder smooth muscle (SM) cells results in profound alterations of the histomorphometry, phenotype, and function of the cells. The onset of this process is characterized by the activation of a cascade of signaling events coupled to progressive and, perhaps, interdependent changes of gene expression. In particular, externally applied cyclic stretch to cultured bladder SM cells results in the transient expression of the Cyr61 gene that encodes a cysteine-rich heparin-binding protein originally described as a proangiogenic factor capable of altering the gene programs for angiogenesis, adhesion, and extracellular matrix synthesis. In this study, we investigated the effects of mechanical stretch-induced Cyr61 on the expression of potential mechanosensitive Cyr61 target genes and the signaling pathways involved. We showed that suppression of Cyr61 expression with an adenoviral vector encoding an antisense oligonucleotide reduced mechanical strain-induced VEGF, αv-integrin, and SM α-actin gene expression but had no effect on the myosin heavy chain isoforms SM-1 and SM-2. Signaling pathways involving RhoA GTPase, phosphatidyl inositol 3-kinase, and cytoskeletal actin dynamics altered stretch-induced Cyr61 and Cyr61 target genes. Reciprocally, adenovirus-mediated overexpression of Cyr61 in cells cultured under static conditions increased the expression of VEGF, αv-integrin, and SM α-actin, as well as that of SM-1 and SM-2 isoforms, suggesting that the effects of a sustained expression of Cyr61 extend to SM specific contractile function. These effects were dependent on integrity of the actin cytoskeleton. Together, these results indicate that Cyr61 is an important determinant of the genetic reprogramming that occurs in mechanically challenged cells.

Shear stress induces hepatocyte PAI-1 gene expression through cooperative Sp1/Ets-1 activation of transcription

2006 ◽  
Vol 291 (1) ◽  
pp. G26-G34 ◽  
Author(s):  
Hideki Nakatsuka ◽  
Takaaki Sokabe ◽  
Kimiko Yamamoto ◽  
Yoshinobu Sato ◽  
Katsuyoshi Hatakeyama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Shear Stress ◽  
Consensus Sequence ◽  
Early Gene ◽  
Immediate Early ◽  
Mrna Levels ◽  
Consensus Sequences ◽  
Static Conditions ◽  
Stress Dependent ◽  
Pai 1

Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAI-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5′-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1.

scholarly journals Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediate-early RNA.

1996 ◽  
Vol 40 (9) ◽  
pp. 2004-2011 ◽  
Author(s):  
K P Anderson ◽  
M C Fox ◽  
V Brown-Driver ◽  
M J Martin ◽  
R F Azad
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Antiviral Activity ◽  
Protein Expression ◽  
Human Cytomegalovirus ◽  
Host Cells ◽  
Early Gene ◽  
Immediate Early ◽  
Mrna Levels ◽  
Virus Adsorption

ISIS 2922 is a phosphorothioate oligonucleotide that is complementary to human cytomegalovirus (CMV) immediate-early (IE) RNA and that exhibits potent and specific antiviral activity against CMV in cell culture assays. Specific assay systems were developed to separately characterize the antisense and nonantisense components of the antiviral activity mediated by ISIS 2922. In U373 cells transformed with cDNA encoding the CMV IE 55-kDa (IE55) protein, expression was inhibited at nanomolar concentrations comparable to effective concentrations in antiviral assays. The specificity of inhibition was demonstrated by using control oligonucleotides incorporating progressive base changes to destabilize oligonucleotide-RNA base pairing and by showing a lack of inhibition of the CMV IE72 product expressed from the same promoter. Inhibition of IE55 protein expression correlated with a reduction in mRNA levels consistent with an RNase H-mediated termination event. Studies with virus-infected cells demonstrated that antisense and nonantisense mechanisms contribute to the antiviral activity of ISIS 2922. Base complementarity to target RNA was important for optimal activity in antiviral assays, but base changes affecting parameters other than hybridization affinity also influenced antiviral activity. Sequence-independent inhibition of virus adsorption to host cells by phosphorothioate oligonucleotides was also observed at high concentrations. Therefore, at least three different mechanisms may contribute to the antiviral activity of ISIS 2922 in cell culture: antisense-mediated inhibition of target gene expression; nonantisense, sequence-dependent inhibition of virus replication; and sequence-independent inhibition of virus adsorption to host cells.

scholarly journals Effects of Adeno-Associated Virus on Adenovirus Replication and Gene Expression during Coinfection

2006 ◽  
Vol 80 (16) ◽  
pp. 7807-7815 ◽  
Author(s):  
Jennifer M. Timpe ◽  
Kristin C. Verrill ◽  
James P. Trempe
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Virus Production ◽  
Dna Amplification ◽  
Early Gene ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Adeno Associated Virus ◽  
Transcript Levels

ABSTRACT Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

GATA-6 mediates human bladder smooth muscle differentiation: involvement of a novel enhancer element in regulating α-smooth muscle actin gene expression

2007 ◽  
Vol 293 (3) ◽  
pp. C1093-C1102 ◽  
Author(s):  
Akihiro Kanematsu ◽  
Aruna Ramachandran ◽  
Rosalyn M. Adam
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Smooth Muscle Actin ◽  
Muscle Differentiation ◽  
Mrna Levels ◽  
Muscle Actin ◽  
Bladder Smooth Muscle ◽  
Human Bladder ◽  
Enhancer Element ◽  
Smooth Muscle Differentiation

Hollow organs exposed to pathological stimuli undergo phenotypic modulation characterized by altered expression of smooth muscle contractile proteins and loss of normal function. The molecular mechanisms that regulate smooth muscle differentiation, especially in organs other than the vasculature, are poorly understood. In this study, we describe a role for the GATA-6 transcription factor in regulation of human bladder smooth muscle differentiation. Knockdown of endogenous GATA-6 in primary human bladder smooth muscle cells (pBSMC) led to decreased mRNA levels of the differentiation markers α-smooth muscle actin (α-SMA), calponin, and smooth muscle myosin heavy chain. Similar effects were obtained following downregulation of GATA-6 by forskolin-induced elevation of intracellular cAMP levels. Forskolin treatment of pBSMC abolished recruitment of GATA-6 to the α-SMA promoter in vivo and reduced activity of human α-SMA promoter-directed gene expression by >60%. This inhibitory effect was rescued by enforced expression of wild-type GATA-6 but not by a zinc-finger-deleted mutant, GATA-6-ΔZF, which lacks DNA-binding ability. In silico analysis of a region of the human α-SMA promoter, described previously as a transcriptional enhancer, identified a putative GATA-binding site at position −919/−913. Point mutation of this site in SMA-Luc abrogated GATA-6-induced activation of promoter activity. Together, these results provide the first evidence for a functional role for GATA-6 in regulation of bladder smooth muscle differentiation. In addition, these findings demonstrate that GATA-6 regulates human α-SMA expression via a novel regulatory cis element in the α-SMA promoter-enhancer.

scholarly journals Dysregulation of Cyclin E Gene Expression in Human Cytomegalovirus-Infected Cells Requires Viral Early Gene Expression and Is Associated with Changes in the Rb-Related Protein p130

2000 ◽  
Vol 74 (9) ◽  
pp. 4192-4206 ◽  
Author(s):  
Anita K. McElroy ◽  
Roopashree S. Dwarakanath ◽  
Deborah H. Spector
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Human Cytomegalovirus ◽  
Cyclin E ◽  
Early Gene ◽  
Immediate Early ◽  
Mrna Levels ◽  
Early Gene Expression ◽  
Cell Extracts ◽  
Infected Cells

ABSTRACT We have previously shown that many cell cycle regulatory gene products are markedly affected by infection of primary fibroblasts with human cytomegalovirus (HCMV) (F. M. Jault, J. M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697–6704, 1995). One of these proteins, cyclin E, is a key determinant of cell cycle progression during G1, and its mRNA levels are significantly increased in HCMV-infected fibroblasts (B. S. Salvant, E. A. Fortunato, and D. H. Spector, J. Virol. 72:3729–3741, 1998). To determine the molecular basis of this effect, we have examined the events that occur at the endogenous cyclin E promoter during the course of infection. In vivo dimethyl sulfate footprinting of the cyclin E promoter revealed several regions of protection and hypersensitivity that were unique to infected cells. In accord with this observation, we find that the virus-induced cyclin E transcripts initiate downstream of the start site identified in mock-infected cells, in regions where these newly appearing protected and hypersensitive sites occur. Viral gene expression is required for this induction. However, the viral immediate-early proteins IE1-72 and IE2-86, either alone or in combination, cannot induce expression of the endogenous cyclin E. The virus must progress past the immediate-early phase and express an early gene product(s) for activation of cyclin E expression. Moreover, IE1-72 does not appear to be required, as infection of cells with an HCMV mutant containing a deletion in the IE1-72 gene leads to full upregulation of cyclin E expression. Using electrophoretic mobility shift assays with infected cell extracts and a region of the cyclin E promoter that includes two previously defined E2F sites as the probe, we detected the appearance of an infection-specific banding pattern. One of the infection-specific bands contained the proteins E2F-4, DP-1, and p130, which were maintained in the infected cells as uniquely phosphorylated species. These results suggest that an altered E2F-4–DP-1–p130 complex along with viral early gene expression may play a role in the transcriptional regulation of cyclin E mRNA during HCMV infection.

scholarly journals Differential stimulation of sea urchin early and late H2B histone gene expression by a gastrula nuclear extract after injection into Xenopus laevis oocytes.

1988 ◽  
Vol 8 (3) ◽  
pp. 1236-1246 ◽  
Author(s):  
R Maxson ◽  
M Ito ◽  
S Balcells ◽  
M Thayer ◽  
M French ◽  
...  
Keyword(s):  
Gene Expression ◽  
Xenopus Laevis ◽  
Dna Sequence ◽  
Sea Urchin ◽  
Nuclear Extract ◽  
Early Gene ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Mrna Levels ◽  
Histone Genes

Sea urchin early histone genes are active in preblastula embryos; late histone genes are maximally expressed during subsequent stages of embryogenesis. We used the Xenopus laevis oocyte to assay for trans-acting factors involved in this differential regulation. Sea urchin nuclear proteins were prepared by extracting gastrula-stage chromatin successively with 0.45, 1, and 2 M NaCl. We injected three fractions into oocytes along with plasmids bearing sea urchin early and late H2b histone genes. While neither the 0 to 0.45 M nor the 1 to 2 M salt fraction affected H2b gene expression, the 0.45 to 1 M salt fraction stimulated early and late H2b mRNA levels significantly. Late H2b gene expression was stimulated preferentially when the early and late genes were coinjected into the same oocytes. This extract did not stimulate the accumulation of transcripts of injected herpesvirus thymidine kinase genes or of the sea urchin Spec 1 gene, suggesting that the stimulatory activity is not a general transcription factor. We localized the DNA sequence required for the stimulatory effect to a region of the late H2b gene located between -43 and +62 relative to the transcription start site. A component of the 0.45 to 1 M salt wash fraction specifically bound to the 105-base-pair late gene DNA sequence and to the corresponding early gene fragment. The abundance of this binding activity decreased on a per genome basis during early development of the sea urchin.

scholarly journals Estrogen Opposes the Apoptotic Effects of Bone Morphogenetic Protein 7 on Tissue Remodeling

2000 ◽  
Vol 20 (13) ◽  
pp. 4626-4634 ◽  
Author(s):  
David G. Monroe ◽  
Donald F. Jin ◽  
Michel M. Sanders
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Signaling Pathways ◽  
Bone Morphogenetic Protein ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Bone Morphogenetic Protein 7 ◽  
Reproductive Tissues ◽  
Morphogenetic Protein ◽  
Estrogen Withdrawal

ABSTRACT Interactions between estrogen and growth factor signaling pathways at the level of gene expression play important roles in the function of reproductive tissues. For example, estrogen regulates transforming growth factor beta (TGFβ) in the uterus during the proliferative phase of the mammalian reproductive cycle. Bone morphogenetic protein 7 (BMP-7), a member of the TGFβ superfamily, is also involved in the development and function of reproductive tissues. However, relatively few studies have addressed the expression of BMP-7 in reproductive tissues, and the role of BMP-7 remains unclear. As part of an ongoing effort to understand how estrogen represses gene expression and to study its interactions with other signaling pathways, chick BMP-7 (cBMP-7) was cloned. cBMP-7 mRNA levels are repressed threefold within 8 h following estrogen treatment in the chick oviduct, an extremely estrogen-responsive reproductive tissue. This regulation occurs at the transcriptional level. Estrogen has a protective role in many tissues, and withdrawal from estrogen often leads to tissue regression; however, the mechanisms mediating regression of the oviduct remain unknown. Terminal transferase-mediated end-labeling and DNA laddering assays demonstrated that regression of the oviduct during estrogen withdrawal involves apoptosis, which is a novel observation. cBMP-7 mRNA levels during estrogen withdrawal increase concurrently with the apoptotic index of the oviduct. Furthermore, addition of purified BMP-7 induces apoptosis in primary oviduct cells. This report demonstrates that the function of BMP-7 in the oviduct involves the induction of apoptosis and that estrogen plays an important role in opposing this function.

Sign in / Sign up

Export Citation Format

Share Document