scholarly journals Gastrin induces c-fosgene transcription via multiple signaling pathways

1999 ◽  
Vol 276 (2) ◽  
pp. G415-G424 ◽  
Author(s):  
Vinzenz M. Stepan ◽  
Makoto Tatewaki ◽  
Masashi Matsushima ◽  
Chris J. Dickinson ◽  
John del Valle ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Luciferase Activity ◽  
Specific Inhibitor ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Response Element ◽  
Gf 109203X ◽  
Fold Induction ◽  
Rho A

We previously observed that the trophic actions of gastrin (G17) on the AR42J rat acinar cell line are mediated by mitogen-activated protein kinase (MAPK)-induced c- fos gene transcription via protein kinase C (PKC)-dependent and -independent pathways. In this study, we further investigated the signaling pathways that target c- fos in response to G17. G17 led to a sixfold induction in luciferase activity in cells transfected with plasmids containing the −356+109 sequence of the murine c- fos promoter, which includes the Sis-inducible element (SIE), serum response element (SRE), and the Ca2+/cAMP response element (CRE) regulatory elements. Addition of either the selective PKC inhibitor GF-109203X or the MAPK/extracellular signal-regulated kinase inhibitor PD-98059 resulted in an 80% reduction in luciferase activity. G17 induced the transcriptional activity of both Elk-1 and Sap-1a, transcription factors that bind to the E26 transformation specific (Ets) DNA sequence of the SRE, and this effect was inhibited by both GF-109203X and PD-98059. Point mutations in the Ets sequence led to a 4-fold induction of c- fos transcription stimulated by G17 and to a 1.3-fold induction in response to epidermal growth factor (EGF). In contrast, mutations in the CA rich G (CArG) sequence of the SRE prevented transcriptional activation by both G17 and EGF. G17 induction of the Ets mutant construct was unaffected by either GF-109203X or PD-98059. Because activation of the SRE involves the small GTP-binding protein Rho A, we examined the role of Rho A in G17 induction of c- fos transcription. Inactivation of Rho A by either the specific inhibitor C3 or by expression of a dominant negative Rho A gene inhibited G17 induction of both the wild-type and the Ets mutant constructs by 60%. C3 also inhibited G17-stimulated AR42J cell proliferation. Thus G17 targets the c- fos promoter CArG sequence via Rho A-dependent pathways, and Rho A appears to play an important role in the regulation of the trophic action of G17.

scholarly journals Essential role of protein kinase C ζ in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP

2007 ◽  
Vol 176 (7) ◽  
pp. 1049-1060 ◽  
Author(s):  
Kageaki Kuribayashi ◽  
Kiminori Nakamura ◽  
Maki Tanaka ◽  
Tsutomu Sato ◽  
Junji Kato ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Morphological Changes ◽  
Specific Inhibitor ◽  
Squamous Carcinoma ◽  
Inflammatory Cells ◽  
Dominant Negative ◽  
Chemotactic Peptide ◽  
Kinase C ◽  
Pkc Ζ

Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) ζ was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKCζ in SASH1 cells, myristoylated PKCζ peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway.

HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway

2021 ◽  
Author(s):  
Toshihiro Kobayashi ◽  
Hitomi Imachi ◽  
Kensaku Fukunaga ◽  
Jingya Lyu ◽  
Seisuke Sato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Promoter Activity ◽  
Active Form ◽  
Specific Inhibitor ◽  
Density Lipoprotein ◽  
Dominant Negative ◽  
Activation Mechanism ◽  
Dominant Negative Mutant ◽  
Type I

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating adiponectin is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of adiponectin through the Ca²+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. Adiponectin expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on adiponectin promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also cancelled the effect of HDL on APN gene expression. These results suggest that HDL has important role to improve the function of adipocytes by activating hSR-BI/CLA-1 and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

scholarly journals Transforming Growth Factor β-Activated Kinase 1 Is a Key Mediator of Ovine Follicle-Stimulating Hormone β-Subunit Expression

Endocrinology ◽  
2005 ◽  
Vol 146 (11) ◽  
pp. 4814-4824 ◽  
Author(s):  
Nedal Safwat ◽  
Jun Ninomiya-Tsuji ◽  
A. Jesse Gore ◽  
William L. Miller
Keyword(s):  
P38 Mapk ◽  
Transforming Growth Factor ◽  
Specific Inhibitor ◽  
Transforming Growth Factor Β ◽  
Cell Types ◽  
Dominant Negative ◽  
Β Subunit ◽  
Subunit Expression ◽  
Fold Induction ◽  
Tgfβ Superfamily

FSH, a key regulator of gonadal function, contains a β-subunit (FSHβ) that is transcriptionally induced by activin, a member of the TGFβ-superfamily. This study used 4.7 kb of the ovine FSHβ-promoter linked to luciferase (oFSHβLuc) plus a well-characterized activin-responsive construct, p3TPLuc, to investigate the hypothesis that Smad3, TGFβ-activated kinase 1 (TAK1), or both cause activin-mediated induction of FSH. Overexpression of either Smad3 or TAK1 induced oFSHβLuc in gonadotrope-derived LβT2 cells as much as activin itself. Induction of p3TPLuc by activin is known to require Smad3 activation in many cell types, and this was true in LβT2 cells, where 10-fold induction by activin (2–8 h after activin treatment) was blocked more than 90% by two dominant negative (DN) inhibitors of Smad3 [DN-Smad3 (3SA) and DN-Smad3 (D407E)]. By contrast, 6.5-fold induction of oFSHβLuc by activin (10–24 h after activin treatment) was not blocked by either DN-Smad inhibitor, suggesting that activation of Smad3 did not trigger induction of oFSHβLuc. By contrast, inhibition of TAK1 by a DN-TAK1 construct led to a 50% decrease in activin-mediated induction of oFSHβLuc, and a specific inhibitor of TAK1 (5Z-7-Oxozeanol) blocked induction by 100%, indicating that TAK1 is necessary for activin induction of oFSHβLuc. Finally, inhibiting p38-MAPK (often activated by TAK1) blocked induction of oFSHβLuc by 60%. In conclusion, the data presented here indicate that activation of TAK1 (and probably p38-MAPK), but not Smad3, is necessary for triggering induction of oFSHβ by activin.

scholarly journals Akt-Dependent Phosphorylation Specifically Regulates Cot Induction of NF-κB-Dependent Transcription

2002 ◽  
Vol 22 (16) ◽  
pp. 5962-5974 ◽  
Author(s):  
Lawrence P. Kane ◽  
Marianne N. Mollenauer ◽  
Zheng Xu ◽  
Christoph W. Turck ◽  
Arthur Weiss
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Cellular Transformation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Iκb Kinase ◽  
Ikk Complex ◽  
Mitogen Activated Protein ◽  
Tumor Necrosis Factor Induction ◽  
Necrosis Factor

ABSTRACT The Akt (or protein kinase B) and Cot (or Tpl-2) serine/threonine kinases are associated with cellular transformation. These kinases have also been implicated in the induction of NF-κB-dependent transcription. As a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, Cot can also activate MAP kinase signaling pathways that target AP-1 and NFAT family transcription factors. Here we show that Akt and Cot physically associate and functionally cooperate. Akt appears to function upstream of Cot, as Akt can enhance Cot induction of NF-κB-dependent transcription, and dominant-negative Cot blocks the activation of this element by Akt. Furthermore, deletion analysis shows that binding to Akt is critical for Cot function. The regulation of NF-κB-dependent transcription by Cot requires Akt-dependent phosphorylation of serine 400 (S400), near the carboxy terminus of Cot. However, phosphorylation at this site is not required for Cot kinase activity or AP-1 induction, suggesting it specifically regulates Cot effector function at the level of the NF-κB pathway. Mutation of S400 in Cot does indeed abolish its ability to activate IκB-kinase (IKK) complexes, but paradoxically it allows for increased Cot association with the IKK complex. This mutated form of Cot also acts as a dominant negative for T-cell antigen receptor/CD28- or Akt/phorbol myristate acetate-induced NF-κB induction, while having relatively little effect on tumor necrosis factor induction of NF-κB. These findings suggest that the activation of different signaling pathways by MAP3Ks may be regulated separately and may provide evidence for how such discrimination by one member of this kinase family occurs.

scholarly journals Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins.

1995 ◽  
Vol 15 (12) ◽  
pp. 6829-6837 ◽  
Author(s):  
M Tanaka ◽  
R Gupta ◽  
B J Mayer
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Erk Activation ◽  
Mitogen Activated Protein

SH2/SH3 adapters are thought to function in signal transduction pathways by coupling inputs from tyrosine kinases to downstream effectors such as Ras. Members of the mitogen-activated protein kinase family are known to be activated by a variety of mitogenic stimuli, including tyrosine kinases such as Abl and the epidermal growth factor (EGF) receptor. We have used activation of the mitogen-activated protein kinase Erk-1 as a model system with which to examine whether various dominant-negative SH2/SH3 adapters (Grb2, Crk, and Nck) could block signaling pathways leading to Erk activation. Activation of Erk-1 by oncogenic Abl was effectively inhibited by Grb2 with mutations in either its SH2 or SH3 domain or by Crk-1 with an SH3 domain mutation. The Crk-1 SH2 mutant was less effective, while Nck SH2 and SH3 mutants had little or no effect on Erk activation. These results suggest that both Crk and Grb2 may contribute to the activation of Erk by oncogenic Abl, whereas Nck is unlikely to participate in this pathway. Next we examined whether combinations of these dominant-negative adapters could inhibit Erk activation more effectively than each mutant alone. When combinations of Crk-1 and Grb2 mutants were analyzed, the combination of the Crk-1 SH3 mutant plus the Grb2 SH3 mutant gave a striking synergistic effect. This finding suggests that in Abl-transformed cells, more than one class of tyrosine-phosphorylated sites (those that bind the Grb2 SH2 domain and those that bind the Crk SH2 domain) can lead to Ras activation. In contrast to results with Abl, Erk activation by EGF was strongly inhibited only by Grb2 mutants; Crk and Nck mutants had little or no effect. This finding suggests that Grb2 is the only adapter involved in the activation of Erk by EGF. Dominant-negative adaptors provide a novel means to identify binding interactions important in vivo for signaling in response to a variety of stimuli.

scholarly journals β2-adrenoceptor Activation Stimulates IL-6 Production via PKA, ERK1/2, Src, and Beta-arrestin2 Signaling Pathways in Human Bronchial Epithelia

2021 ◽  
Author(s):  
Rui Gang Zhang ◽  
Ya Niu ◽  
Ke Wu Pan ◽  
Hao Pang ◽  
Chung Ling Chen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Specific Inhibitor ◽  
Intracellular Camp ◽  
Cytokine Release ◽  
High Concentration ◽  
Airway Epithelia ◽  
Bronchial Epithelia ◽  
Adrenoceptor Agonists ◽  
Camp Receptor

Abstract Background: β 2 -adrenoceptor agonists are widely used to treat asthma because of their bronchial-dilation effects. However, a recent study describing a side effect of aggravating eosinophilic inflammation in the mouse airway epithelia by β 2 -adrenoceptor agonists could impact the future clinical use of these bronchodilators. We previously reported that isoprenaline, via the apical and basolateral β 2 -adrenoceptor, induced Cl - secretion by activating cyclic AMP (cAMP)-dependent pathways in human bronchial epithelia. Despite these results, whether and how the β 2 -adrenoceptor-mediated cAMP-dependent pathway contributes to pro-inflammatory cytokine release in human bronchial epithelia remains poorly understood.Methods: We investigated β 2 -adrenoceptor-mediated signaling pathways involved in the production of two pro-inflammatory cytokines, interleukin (IL)-6 and IL-8, in 16HBE14o- human bronchial epithelia. The effects of isoprenaline or formoterol were assessed in the presence of protein kinase A (PKA), exchange protein directly activated by cAMP (EPAC), Src, and extracellular signal-regulated protein kinase (ERK)1/2 inhibitors. The involvement of b-arrestin2 was examined using siRNA knockdown. Results: Both isoprenaline and formoterol (both β 2 agonists) induced IL-6, but not IL-8, release, which could be inhibited by ICI 118551 (β 2 antagonist). The PKA-specific inhibitor, H89, partially inhibited IL-6 release. Another intracellular cAMP receptor, EPAC, was not involved in IL-6 release. Isoprenaline-mediated IL-6 secretion was attenuated by dasatinib, a Src inhibitor, and PD98059, an ERK1/2 inhibitor. Isoprenaline treatment also led to ERK1/2 phosphorylation. In addition, knockdown of β-arrestin2 by siRNA specifically suppressed cytokine release when a high concentration of isoprenaline (1 mM) was used. Conclusion: Our results suggest that activation of the β 2 -adrenoceptor in 16HBE14o- cells stimulated the PKA/Src/ERK1/2 and/or β-arrestin2 signaling pathways, leading to IL-6 release. Therefore, our data reveal that β 2 -adrenoceptor signaling plays a role in the immune regulation of human airway epithelia.

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.

Interactions Between the Inositol 1,4,5-Trisphosphate and Cyclic AMP Signaling Pathways Regulate Larval Molting in Drosophila

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 309-318 ◽  
Author(s):  
K Venkatesh ◽  
G Siddhartha ◽  
Rohit Joshi ◽  
Sonal Patel ◽  
Gaiti Hasan
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Signaling Pathways ◽  
Feedback Inhibition ◽  
Receptor Gene ◽  
Dominant Negative ◽  
Neural Signals ◽  
Inositol 1,4,5 Trisphosphate ◽  
Kinase A ◽  
Insp3 Receptor

Abstract Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. In this study we have analyzed, using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting in Drosophila. Previous studies have shown that mutants for the inositol 1,4,5-trisphosphate receptor gene (itpr) are larval lethals. In addition they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Here we show that mutants for adenylate cyclase (rut) synergize, during larval molting, with itpr mutant alleles, indicating that both cAMP and InsP3 signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP3 receptor. Furthermore, our studies predict the existence of feedback inhibition through protein kinase A on the InsP3 receptor by increased levels of 20-hydroxyecdysone.

scholarly journals Calcitonin stimulates expression of the rat 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter in HEK-293 cells expressing calcitonin receptor: identification of signaling pathways

2004 ◽  
Vol 32 (1) ◽  
pp. 87-98 ◽  
Author(s):  
XH Gao ◽  
PP Dwivedi ◽  
JL Omdahl ◽  
HA Morris ◽  
BK May
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Basal Expression ◽  
293 Cells ◽  
Gc Box

Regulation of the gene for renal 25-hydroxyvitamin D-24-hydroxylase (CYP24) is important for controlling the level of circulating 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We report here for the first time that the peptide hormone calcitonin significantly stimulates expression of a rat CYP24 promoter-luciferase construct in both transiently and stably transfected kidney HEK-293 cells. A GC box at -114/-101 and a CCAAT box at -62/-51 have been identified that underlie both basal expression of the CYP24 promoter and the calcitonin inductive response. Data from overexpression studies suggested that Sp1 and NF-Y are the proteins that function through the GC and CCAAT boxes respectively. ERK1/2 signaling pathways were not involved in the calcitonin-mediated response, since stimulation of the promoter was unaffected by the pharmacological ERK1/2 inhibitor PD98059 and by a dominant negative mutant of ERK1/2 (ERK1K71R). In contrast, calcitonin induction but not basal expression was dependent on protein kinase A and protein kinase C (PKC) activities with the inhibitors H89 and calphostin C lowering induction by 50-60%. The atypical PKC, PKCzeta contributes to calcitonin induction, but not to basal expression of the CYP24 promoter, since overexpression of a dominant negative clone PKCzetaK281 M lowered induction by 50%. Cotransfection of a dominant negative form of Ras resulted in calcitonin-mediated induction being reduced also by about 50%. A Ras-PKCzeta signaling pathway for calcitonin action is proposed, which acts through the GC box. The findings have been extrapolated to the in vivo situation where we suggest that induction of renal CYP24 by calcitonin could be important under hypercalcemic conditions thus contributing to the lowering of circulating 1,25(OH)2D3 levels.

scholarly journals Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase promoter contains a CREB binding site that regulates cAMP action in Caco-2 cells

2000 ◽  
Vol 345 (2) ◽  
pp. 201-206 ◽  
Author(s):  
Anke EGGERS ◽  
Concha CAUDEVILLA ◽  
Guillermina ASINS ◽  
Fausto G. HEGARDT ◽  
Dolors SERRA
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Camp Response Element Binding ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mrna Levels ◽  
Camp Response Element ◽  
Response Element ◽  
Kinase A

cAMP increases transcription of the mitochondrial (mit.) gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase, which encodes an enzyme that has been proposed as a control site of ketogenesis. The incubation of Caco-2 cells with cAMP increased mit.HMG-CoA synthase mRNA levels 4-fold within 24 h. We have identified an active cAMP-response element (CRE) located 546 bp upstream of the mit.HMG-CoA synthase promoter that is necessary for the induction of expression by dibutyryl cAMP. Co-transfections of constructs, containing the CRE element of the mit.HMG-CoA synthase promoter fused to the gene for chloramphenicol acetyltransferase, with protein kinase A and a dominant-negative mutant of cAMP-response-element-binding protein (CREB) show that the response to cAMP is mediated by the transcription factor CREB. The CRE element confers responsiveness of protein kinase A to a heterologous promoter in transfection assays in Caco-2 cells. Gel-retardation assays revealed that the mit.HMG-CoA synthase CRE binds to recombinant CREB. The shifted band obtained with the putative mit.HMG-CoA synthase CRE sequence and nuclear proteins from Caco-2 cells competed with CRE sequences of other genes such as somatostatin and phosphoenolpyruvate carboxykinase. We conclude that the regulation of the expression of the gene for mit.HMG-CoA synthase in Caco-2 cells by cAMP is mediated by a CRE sequence in the promoter.

Sign in / Sign up

Export Citation Format

Share Document