Effects of the JNK inhibitor anthra[1,9-cd]pyrazol-6(2H)-one (SP-600125) on soluble guanylyl cyclase α1gene regulation and cGMP synthesis

2005 ◽  
Vol 289 (4) ◽  
pp. C778-C784 ◽  
Author(s):  
Joshua S. Krumenacker ◽  
Alexander Kots ◽  
Ferid Murad
Keyword(s):  
Pc12 Cells ◽  
Guanylyl Cyclase ◽  
Soluble Guanylyl Cyclase ◽  
Fetal Lung ◽  
Cell Types ◽  
Mrna Levels ◽  
Disease States ◽  
Jun Kinase ◽  
Constitutively Active

The decreased expression of the nitric oxide (NO) receptor, soluble guanylyl cyclase (sGC), occurs in response to multiple stimuli in vivo and in cell culture and correlates with various disease states such as hypertension, inflammation, and neurodegenerative disorders. The ability to understand and modulate sGC expression and cGMP levels in any of these conditions could be a valuable therapeutic tool. We demonstrate herein that the c-Jun NH2-terminal kinase JNK II inhibitor anthra[1,9- cd]pyrazol-6(2 H)-one (SP-600125) completely blocked the decreased expression of sGCα1-subunit mRNA by nerve growth factor (NGF) in PC12 cells. Inhibitors of the ERK and p38 MAPK pathways, PD-98059 and SB-203580, had no effect. SP-600125 also inhibited the NGF-mediated decrease in the expression of sGCα1protein as well as sGC activity in PC12 cells. Other experiments revealed that decreased sGCα1mRNA expression through a cAMP-mediated pathway, using forskolin, was not blocked by SP-600125. We also demonstrate that TNF-α/IL-1β stimulation of rat fetal lung (RFL-6) fibroblast cells resulted in sGCα1mRNA inhibition, which was blocked by SP-600125. Expression of a constitutively active JNKK2-JNK1 fusion protein in RFL-6 cells caused endogenous sGCα1mRNA levels to decrease, while a constitutively active ERK2 protein had no effect. Collectively, these data demonstrate that SP-600125 may influence the intracellular levels of the sGCα1-subunit in certain cell types and may implicate a role for c-Jun kinase in the regulation of sGCα1expression.

scholarly journals The Expression Pattern of Nitric Oxide-sensitive Guanylyl Cyclase in the Rat Heart Changes During Postnatal Development

2002 ◽  
Vol 50 (10) ◽  
pp. 1325-1331 ◽  
Author(s):  
Sönke Behrends ◽  
Andrea Mietens ◽  
Jörg Kempfert ◽  
Markus Koglin ◽  
Hasso Scholz ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Postnatal Development ◽  
Guanylyl Cyclase ◽  
Rat Heart ◽  
Constitutive Expression ◽  
Soluble Guanylyl Cyclase ◽  
Cell Types ◽  
Perinatal Period ◽  
Dynamic Regulation ◽  
Subsequent Decrease

Nitric oxide (NO)-releasing drugs such as glyceryl trinitrate have been used in the treatment of ischemic heart disease for more than a century. Nevertheless, a detailed analysis of the expression of the NO target enzyme soluble guanylyl cyclase (sGC) in the heart is missing. The aim of the current study was to elucidate the expression, cell distribution, and activity of sGC in the rat heart during postnatal development. Using a novel antibody raised against a C-terminal peptide of the rat β1-subunit of sGC, the enzyme was demonstrated in early postnatal and adult hearts by Western blotting analyses, showing maximal expression in 10-day-old animals. Measurements of basal, NO-, and NO/YC-1-stimulated sGC activity revealed an increase of sGC activity in hearts from neonatal to 10-day-old rats, followed by a subsequent decrease in adult animals. As shown by immunohis-tochemical analysis, sGC expression was present in vascular endothelium and smooth muscle cells in neonatal heart but expression shifted to endothelial cells in adult animals. In isolated cardiomyocytes, sGC activity was not detectable under basal conditions but significant sGC activity could be detected in the presence of NO. An increase in expression during the perinatal period and changes in the cell types expressing sGC at different phases of development suggest dynamic regulation rather than constitutive expression of the NO receptor in the heart.

Cytokine-induced changes in chromatin structure and in vivo footprints in the inducible NOS promoter

2001 ◽  
Vol 280 (3) ◽  
pp. L390-L399 ◽  
Author(s):  
Jane K. Mellott ◽  
Harry S. Nick ◽  
Michael F. Waters ◽  
Timothy R. Billiar ◽  
David A. Geller ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Specific Pattern ◽  
Mrna Levels ◽  
Inos Gene ◽  
In Vivo Footprinting ◽  
Induced Changes

Transcription of the human inducible nitric oxide synthase ( iNOS) gene is regulated by inflammatory cytokines in a tissue-specific manner. To determine whether differences in cytokine-induced mRNA levels between pulmonary epithelial cells (A549) and hepatic biliary epithelial cells (AKN-1) result from different protein or DNA regulatory mechanisms, we identified cytokine-induced changes in DNase I-hypersensitive (HS) sites in 13 kb of the iNOS 5′-flanking region. Data showed both constitutive and inducible HS sites in an overlapping yet cell type-specific pattern. Using in vivo footprinting and ligation-mediated PCR to detect potential DNA or protein interactions, we examined one promoter region near −5 kb containing both constitutive and cytokine-induced HS sites. In both cell types, three in vivo footprints were present in both control and cytokine-treated cells, and each mapped within a constitutive HS site. The remaining footprint appeared only in response to cytokine treatment and mapped to an inducible HS site. These studies, performed on chromatin in situ, identify a portion of the molecular mechanisms regulating transcription of the human iNOS gene in both lung- and liver-derived epithelial cells.

Glucocorticoids upregulate tropoelastin expression during late stages of fetal lung development

1995 ◽  
Vol 268 (3) ◽  
pp. L491-L500 ◽  
Author(s):  
R. A. Pierce ◽  
W. I. Mariencheck ◽  
S. Sandefur ◽  
E. C. Crouch ◽  
W. C. Parks
Keyword(s):  
Lung Development ◽  
Matrix Protein ◽  
Fetal Lung ◽  
Extracellular Matrix Protein ◽  
Transferase Activity ◽  
Mrna Levels ◽  
Pregnant Rats ◽  
Fetal Lung Development ◽  
Chloramphenicol Acetyl Transferase Activity

The production of elastin, an essential extracellular matrix protein of terminal airway interstitium, occurs mostly during early development. Because glucocorticoids influence airway maturation, we studied the effect of dexamethasone (Dex) on tropoelastin expression during fetal lung development. Timed-pregnant rats were treated with Dex (1 mg/kg daily), and fetal lungs were collected 3 days later at 17, 19, and 21 days of gestation. Dex treatment resulted in about a threefold increase in tropoelastin mRNA levels at 19 days concomitant with accelerated airway development. By in situ hybridization, Dex treatment increased the number of tropoelastin-expressing cells and the level of tropoelastin mRNA per cell. In organ culture, Dex increased lung tropoelastin expression and augmented cortisol stimulation of tropoelastin expression. In fetal pulmonary artery smooth muscle cells, 10(-8) M Dex upregulated tropoelastin mRNA expression and increased tropoelastin promoter-chloramphenicol acetyl transferase activity in transient transfections. These data indicate that pharmacologically administered glucocorticoids transcriptionally upregulate fetal lung tropoelastin expression and suggest that steroid hormones may be important regulators of elastin production in vivo.

scholarly journals cGMP: a unique 2nd messenger molecule – recent developments in cGMP research and development

2019 ◽  
Vol 393 (2) ◽  
pp. 287-302 ◽  
Author(s):  
Andreas Friebe ◽  
Peter Sandner ◽  
Achim Schmidtko
Keyword(s):  
Guanylyl Cyclase ◽  
Soluble Guanylyl Cyclase ◽  
Cell Types ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Therapeutic Implications ◽  
Cellular Effects ◽  
Recent Developments ◽  
And Function ◽  
Messenger Molecule

AbstractCyclic guanosine monophosphate (cGMP) is a unique second messenger molecule formed in different cell types and tissues. cGMP targets a variety of downstream effector molecules and, thus, elicits a very broad variety of cellular effects. Its production is triggered by stimulation of either soluble guanylyl cyclase (sGC) or particulate guanylyl cyclase (pGC); both enzymes exist in different isoforms. cGMP-induced effects are regulated by endogenous receptor ligands such as nitric oxide (NO) and natriuretic peptides (NPs). Depending on the distribution of sGC and pGC and the formation of ligands, this pathway regulates not only the cardiovascular system but also the kidney, lung, liver, and brain function; in addition, the cGMP pathway is involved in the pathogenesis of fibrosis, inflammation, or neurodegeneration and may also play a role in infectious diseases such as malaria. Moreover, new pharmacological approaches are being developed which target sGC- and pGC-dependent pathways for the treatment of various diseases. Therefore, it is of key interest to understand this pathway from scratch, beginning with the molecular basis of cGMP generation, the structure and function of both guanylyl cyclases and cGMP downstream targets; research efforts also focus on the subsequent signaling cascades, their potential crosstalk, and also the translational and, ultimately, the clinical implications of cGMP modulation. This review tries to summarize the contributions to the “9th International cGMP Conference on cGMP Generators, Effectors and Therapeutic Implications” held in Mainz in 2019. Presented data will be discussed and extended also in light of recent landmark findings and ongoing activities in the field of preclinical and clinical cGMP research.

Cytokine-mediated PGE2expression in human colonic fibroblasts

1998 ◽  
Vol 275 (4) ◽  
pp. C988-C994 ◽  
Author(s):  
Edward C. Kim ◽  
Yingting Zhu ◽  
Valerie Andersen ◽  
Daniela Sciaky ◽  
H. James Cao ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Shape Change ◽  
Cell Types ◽  
Mrna Levels ◽  
Primary Fibroblast ◽  
Fibroblast Cultures ◽  
Epithelial Cell Lines ◽  
Factor Α

We investigated prostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84, HT-29, and LS 174T) and the effect of PGE2 on fibroblast morphology. Cytokine-stimulated PGE2production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) protein and mRNA expression were evaluated. Basal PGE2 levels were low in all cell types (0.15–6.47 ng/mg protein). Treatment for 24 h with interleukin-1β (IL-1β; 10 ng/ml) or tumor necrosis factor-α (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions of PGE2 synthesis in CCD-18Co cultures and similar results in primary fibroblast cultures; maximal inductions with IL-1β in colonic epithelial cell lines were from zero to fivefold. Treatment of CCD-18Co fibroblasts with IL-1β caused maximal 21- and 53-fold increases, respectively, in PGHS-2 protein and mRNA levels without altering PGHS-1 expression. PGE2 (0.1 μmol/l) elicited a dramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and target for colonic prostanoids in vivo.

Cellular distribution of insulin-like growth factor binding protein mRNAs and peptides during rat lung development

1997 ◽  
Vol 155 (2) ◽  
pp. 313-327 ◽  
Author(s):  
LD Wallen ◽  
W Myint ◽  
K Nygard ◽  
S Shimasaki ◽  
DR Clemmons ◽  
...  
Keyword(s):  
Airway Epithelium ◽  
Lung Development ◽  
Fetal Lung ◽  
Northern Analysis ◽  
Postnatal Life ◽  
Mrna Levels ◽  
Igf Binding Proteins ◽  
Paracrine Factors ◽  
Igfbp 3

A role for IGF binding proteins (IGFBPs) in lung development is suggested by the identification of IGFBPs in lung tissue and production of IGFBPs by fetal lung cells in culture. To characterize the expression of IGFBPs during lung development in the rat in vivo (16 days gestation through adulthood), the expression of IGFBP mRNAs (IGFBP-1 to IGFBP-6) was examined by Northern analysis and in situ hybridization, and IGFBP peptides (IGFBP-2, IGFBP-3, and IGFBP-5) were localized by immunohistochemistry. IGFBP-1 mRNA was not detectable. IGFBP-2 mRNA (1.8 kb) was expressed in both fetal and postnatal life with peak expression during the fetal pseudoglandular stage. IGFBP-2 mRNA was localized mainly to airway epithelium. IGFBP-3 mRNA (2.4 kb) was maximally expressed postnatally in the saccular stage of lung development; it was identified in airway epithelium and interstitium in the fetal lung, but predominantly in airway epithelium after birth. IGFBP-4 (2.6 kb) and IGFBP-5 (6.0 kb) mRNA levels were maximal after birth, from 3 to 21 days postnatal (saccular and alveolar stage). IGFBP-4 mRNA was localized primarily to the interstitium and blood vessels early in development, but was abundant in airway epithelium in the adult. IGFBP-5 mRNA was most abundant in the airway epithelium. IGFBP-3, IGFBP-4, IGFBP-5, and to a lesser extent IGFBP-6 were localized to the large cartilaginous airways in the adult. IGFBP-2, IGFBP-3, and IGFBP-5 peptides were distributed more widely than their respective mRNAs, with a temporal pattern of immunoreactivity following that of their mRNAs. Maximal staining was noted in airway epithelium for IGFBP-2 in the newborn, for IGFBP-3 in the saccular stage (newborn to 3 days postnatal), and for IGFBP-5 in the alveolar stage (5 to 21 days postnatal). Our studies demonstrate that IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 are synthesized and distributed in spatially and temporally different patterns in the developing lung. The widespread distribution of IGFBP immunoreactivity compared with their respective mRNAs suggests that IGFBPs are important paracrine factors in the regulation of IGF action in the developing lung.

scholarly journals The Effect of the Soluble Guanylyl Cyclase Stimulator Olinciguat on ƴ-Globin Gene Induction in K562 Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1078-1078 ◽  
Author(s):  
Joy Miyashiro ◽  
Asha Pant ◽  
Boris Tchernychev ◽  
Todd G Milne ◽  
Mark G Currie ◽  
...  
Keyword(s):  
Cell Line ◽  
Guanylyl Cyclase ◽  
Mode Of Action ◽  
Globin Gene ◽  
Soluble Guanylyl Cyclase ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Mrna Levels ◽  
Globin Mrna ◽  
No Signaling

Abstract Induction of fetal hemoglobin (HbF: α2ƴ2) is a recognized mode of action of hydroxyurea, the sickle cell disease (SCD) standard of care in SCD, and has been shown to prevent red blood cell (RBC) sickling. Discovery of novel HbF inducers is underway and several therapeutics with the potential to increase HbF expression are currently at different stages of preclinical and clinical development. Soluble guanylyl cyclase (sGC) is a heterodimeric heme-containing enzyme whose catalytic activity is regulated by nitric oxide (NO). Binding of NO to heme activates the catalytic domain of sGC, enabling synthesis of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate. sGC stimulators are small molecules that synergize with NO to boost signaling via the NO-sGC-cGMP pathway. This signaling pathway is involved in the regulation of many physiologic processes including inflammation, fibrosis, and blood flow. Perhaps less well-known, cGMP-mediated signaling has also been implicated in the regulation of the gene encoding the ƴ-globin subunit of fetal hemoglobin (Modulation of NO signaling by sGC stimulation, therefore, has the therapeutic potential to target the complex pathology of SCD at multiple levels. In this study, we focused on one potential mode of action of sGC stimulation-increasing HbF expression. We characterized the effects of the sGC stimulator olinciguat on ƴ-globin gene expression. Olinciguat is currently being investigated for the treatment of patients with SCD in a Phase II STRONG-SCD study (NCT03285178). The effect of olinciguat treatment on ƴ-globin mRNA levels was studied in the K562 erythroleukemic cell line. For short-term (8 hours) treatment with olinciguat, K562 cells were maintained in a serum-free media. For long-term (4 and 7 days) treatment, cell culture media contained 1% fetal bovine serum. Hydroxyurea was used as a positive control. Levels of ƴ-globin mRNA were expressed relative to mRNA levels of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. K562 cells were treated for 8 hours with increasing concentrations of olinciguat (0.01, 0.1, 1, and 10 µM). Treatment of K562 cells with 0.1, 1, and 10 µM of olinciguat increased ƴ-globin mRNA levels by 1.43±0.08-, 1.37±0.06-, and 1.47±0.06-fold (mean±SEM), respectively. For comparison, 8 hours of treatment with hydroxyurea (800 µM) increased ƴ-globin mRNA levels by 1.25±0.03-fold. When K562 cells were cultured in the presence of olinciguat for 4 days, significant (P<0.05) induction of ƴ-globin mRNA levels was observed at 1 and 10 µM (1.13±0.03- and 1.55±0.09-fold, respectively). Induction of ƴ-globin mRNA following 4 days of incubation with hydroxyurea (800 µM) was 2.38±0.2-fold. The effects of hydroxyurea and olinciguat on ƴ-globin mRNA levels were compared following 7 days of incubation with the compounds. After 7 days of treatment of K562 cells with 0.1, 1, 3, and 10 µM of olinciguat, ƴ-globin mRNA levels were increased by 1.83±0.19-, 1.66±0.09-, 2.4±0.06-, and 2.9±0.33-fold, respectively. Treatment with 50- and 800-µM hydroxyurea increased levels of ƴ-globin mRNA by 2.33±0.15- and 3.8±0.56-fold, respectively. In conclusion, the sGC stimulator olinciguat increased the expression of mRNA for the ƴ-globin subunit of fetal hemoglobin in the erythroleukemic K562 cell line. This finding indicates that amplifying NO signaling by stimulating sGC may increase HbF expression, thereby preventing pathologic RBC sickling; this extends the potential therapeutic utility of olinciguat in SCD. Finally, the ability of olinciguat to induce HbF in SCD patients will be assessed in the ongoing Phase II STRONG-SCD study (NCT03285178). Disclosures Miyashiro: Ironwood Pharmaceuticals: Employment. Pant:Ironwood Pharmaceuticals: Employment. Tchernychev:Ironwood Pharmaceuticals: Employment, Equity Ownership. Milne:Ironwood Pharmaceutics, Inc: Employment. Currie:Ironwood Pharmaceuticals: Employment. Graul:Ironwood Pharmaceuticals, Inc: Employment. Masferrer:Ironwood Pharmaceuticals, Inc: Employment.

scholarly journals TRAP-based allelic translation efficiency imbalance analysis to identify genetic regulation of ribosome occupancy in specific cell types in vivo

2020 ◽  
Author(s):  
Yating Liu ◽  
Anthony D. Fischer ◽  
Celine L. St. Pierre ◽  
Juan F. Macias-Velasco ◽  
Heather A. Lawson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genetic Regulation ◽  
Transcript Abundance ◽  
Cell Types ◽  
Regulatory Elements ◽  
Model Organisms ◽  
Specific Cell ◽  
Translation Efficiency ◽  
Mrna Levels

AbstractThe alteration of gene expression due to variations in the sequences of transcriptional regulatory elements has been a focus of substantial inquiry in humans and model organisms. However, less is known about the extent to which natural variation contributes to post-transcriptional regulation. Allelic Expression Imbalance (AEI) is a classical approach for studying the association of specific haplotypes with relative changes in transcript abundance. Here, we piloted a new TRAP based approach to associate genetic variation with transcript occupancy on ribosomes in specific cell types, to determine if it will allow examination of Allelic Translation Imbalance (ATI), and Allelic Translation Efficiency Imbalance, using as a test case mouse astrocytes in vivo. We show that most changes of the mRNA levels on ribosomes were reflected in transcript abundance, though ∼1.5% of transcripts have variants that clearly alter loading onto ribosomes orthogonally to transcript levels. These variants were often in conserved residues and altered sequences known to regulate translation such as upstream ORFs, PolyA sites, and predicted miRNA binding sites. Such variants were also common in transcripts showing altered abundance, suggesting some genetic regulation of gene expression may function through post-transcriptional mechanisms. Overall, our work shows that naturally occurring genetic variants can impact ribosome occupancy in astrocytes in vivo and suggests that mechanisms may also play a role in genetic contributions to disease.

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