Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts: a molecular mechanism contributing to organ atrophy

2000 ◽  
Vol 349 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Wei ZHANG ◽  
Catherine E. WATSON ◽  
Connie LIU ◽  
Kevin Jon WILLIAMS ◽  
Victoria P. WERTH
Keyword(s):  
Gene Transcription ◽  
Half Life ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Dermal Fibroblasts ◽  
Steroid Hormone Receptors ◽  
Hyaluronan Synthase ◽  
Osteosarcoma Cell ◽  
Mrna Levels ◽  
In Cells

Glucocorticoid (GC) administration induces atrophy of skin, bone, and other organs, partly by reducing tissue content of glycosaminoglycans, particularly hyaluronic acid (HA). We took advantage of the recent cloning of the three human hyaluronan synthase (HAS) enzymes (HAS1, HAS2 and HAS3), to explore the molecular mechanisms of this side effect. Northern and slot blots performed on RNA extracted from cultured dermal fibroblasts and the MG-63 osteoblast-like osteosarcoma cell line indicated that HAS2 is the predominant HAS mRNA in these cells. Incubation of both cell types for 24 h in the presence of 10-6 M dexamethasone (DEX) resulted in a striking 97-98% suppression of HAS2 mRNA levels. Time-course studies in fibroblasts demonstrated suppression of HAS2 mRNA to 28% of control by 1 h, and to 1.2% of control by 2 h, after addition of DEX. Dose-response studies in fibroblasts indicated that the majority of the suppressive effect required concentrations characteristic of cell-surface GC receptors, a point confirmed by persistent DEX-induced suppression in the presence of RU486, an antagonist of classic cytosolic steroid hormone receptors. Nuclear run-off experiments showed a 70% suppression of HAS2 gene transcription in nuclei from DEX-treated fibroblasts, which is unlikely to fully explain the rapid 50-80-fold reduction in message levels. Experiments with actinomycin D (AMD) demonstrated that the message half-life was 25 min in cells without DEX, whereas the combination of AMD with DEX dramatically increased the half-life of HAS2 mRNA, suggesting that DEX acts by inducing a short-lived destabilizer of the HAS2 message. Direct assessment of HAS2 mRNA stability by wash-out of incorporated uridine label established a half-life of 31 min in cells without DEX, which substantially shortened in the presence of DEX. In conclusion, GCs induce a rapid and sustained, near-total suppression of HAS2 message levels, mediated through substantial decreases in both gene transcription and message stability. These effects may contribute to the loss of HA in GC-treated organs.

Expression of the ornithine decarboxylase gene in response to asparagine in intestinal epithelial cells

1996 ◽  
Vol 271 (1) ◽  
pp. G164-G171 ◽  
Author(s):  
J. Y. Wang ◽  
M. J. Viar ◽  
P. M. Blanner ◽  
L. R. Johnson
Keyword(s):  
Ornithine Decarboxylase ◽  
Half Life ◽  
Cellular Protein ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Normal Rat ◽  
Fetal Bovine ◽  
Mucosal Growth ◽  
Rate Limiting ◽  
In Cells

Refeeding fasted rats significantly stimulates mucosal growth and ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, but the exact mechanism responsible for induction of ODC at the molecular level is unknown. Of normal dietary constituents, the amino acid asparagine markedly increases ODC activity and mucosal growth when administered intragastrically. The current study examined the expression of the ODC gene in IEC-6 cells (a line of normal rat small intestinal crypt cells) after exposure to asparagine. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum. They were deprived of serum for 24 h before experiments. Exposure to asparagine at the dose of 10 mM resulted in the rapid increase in ODC mRNA levels. The increased expression of the ODC gene began 1 h after and peaked between 3 and 5 h after treatment with asparagine. Maximum increases in ODC mRNA levels were approximately fivefold the normal value. Increased levels of ODC mRNA in cells exposed to asparagine were paralleled by increases in ODC protein and enzyme activity and cellular polyamine levels. The half-life of mRNA for ODC in unstimulated IEC-6 cells was approximately 30 min and increased to > 2 h in cells exposed to 10 mM asparagine. The half-life of ODC activity also was increased in asparagine-treated cells. When cellular protein synthesis was inhibited by cycloheximide, asparagine superinduced ODC mRNA levels. Furthermore, asparagine also significantly stimulated DNA synthesis in IEC-6 cells. These results indicate that 1) asparagine stimulates ODC in IEC-6 cells through multiple pathways and 2) increased ODC mRNA levels result partly from a delay in the rate of degradation. These findings suggest that luminal amino acids stimulate gut mucosal growth in association with their ability to regulate ODC gene expression.

Glucocorticoids regulate NHE-3 transcription in OKP cells

1996 ◽  
Vol 270 (1) ◽  
pp. F164-F169 ◽  
Author(s):  
M. Baum ◽  
M. Amemiya ◽  
V. Dwarakanath ◽  
R. J. Alpern ◽  
O. W. Moe
Keyword(s):  
Gene Transcription ◽  
Half Life ◽  
Time Course ◽  
In Vitro Transcription ◽  
Mrna Abundance ◽  
Proton Secretion ◽  
Threefold Increase ◽  
Mrna Half Life ◽  
Synthetic Glucocorticoid

OKP cells express NHE-3, an amiloride-resistant Na+/H+ antiporter, which is likely an isoform responsible for apical proton secretion by the proximal tubule. We have previously shown that an amiloride-resistant Na+/H+ antiporter in OKP cells is regulated by dexamethasone, a synthetic glucocorticoid. The purpose of the present study was to examine the mechanism for the glucocorticoid-mediated increase in Na+/H+ antiporter activity. Incubation of OKP cells with 10(-6) M dexamethasone resulted in a two- to threefold increase in NHE-3 mRNA abundance. This increase was seen after 4 h of incubation with dexamethasone, a time course similar to that found for Na+/H+ antiporter activity. To examine the mechanism for the increase in NHE-3 mRNA abundance, mRNA half-life and in vitro transcription experiments were performed. NHE-3 mRNA had a half-life of 8 h in control and dexamethasone-treated cells. The rate of in vitro transcription was 1.8-fold greater when OKP cells were treated with dexamethasone. These data suggest that the glucocorticoid-mediated increase in Na+/H+ antiporter activity is due to an increase in NHE-3 gene transcription.

scholarly journals Hormonal regulation of stability of glutamine synthetase mRNA in cultured 3T3-L1 adipocytes

1990 ◽  
Vol 267 (1) ◽  
pp. 241-244 ◽  
Author(s):  
K Saini ◽  
P Thomas ◽  
B Bhandari
Keyword(s):  
Gene Expression ◽  
Glutamine Synthetase ◽  
Gene Transcription ◽  
Mrna Stability ◽  
Half Life ◽  
Hormonal Regulation ◽  
Molecular Mechanisms ◽  
Mrna Degradation ◽  
Dibutyryl Cyclic Amp ◽  
Mrna Content

In 3T3-L1 adipocytes, glutamine synthetase (GS; EC 6.3.1.2) is subject to regulation by dexamethasone, insulin and dibutyryl cyclic AMP (Bt2cAMP). Dexamethasone increases GS-mRNA content and GS-gene transcription, whereas insulin and Bt2cAMP prevent these increases. The effects of these modulators on the control of GS-mRNA stability were investigated. We report here that GS mRNA has a half-life of about 110 min. Bt2cAMP increases GS-mRNA degradation by greater than 2-fold (half-life 50 min), whereas insulin or dexamethasone have little effect on GS-mRNA stability. Down-regulation of GS-gene expression by Bt2cAMP will involve a co-ordinate response at the level of gene transcription and mRNA stability. However, the molecular mechanisms by which insulin and dexamethasone regulate GS-gene expression in cultured adipocytes remains to be elucidated.

Synergistic induction of ornithine decarboxylase by asparagine and gut peptides in intestinal crypt cells

1998 ◽  
Vol 274 (6) ◽  
pp. C1476-C1484 ◽  
Author(s):  
Jian-Ying Wang ◽  
Ji Li ◽  
Anami R. Patel ◽  
Stephen Summers ◽  
Li Li ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
Half Life ◽  
Mrna Levels ◽  
Gut Peptides ◽  
Intestinal Crypt ◽  
Simultaneous Addition ◽  
Protein Levels ◽  
Synergistic Induction ◽  
Crypt Cells ◽  
In Cells

The objective of this study was to determine whether the amino acid asparagine stimulated the activity of ornithine decarboxylase (ODC) synergistically with epidermal growth factor (EGF) or gastrin in IEC-6 cells, a line of normal rat small intestinal crypt cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum, and serum was deprived for 24 h before experiments. Exposure to EGF or gastrin alone increased ODC activity 4.5- to 6-fold. Asparagine alone increased the enzyme activity 10- to 13-fold in IEC-6 cells. Simultaneous addition of asparagine and EGF or gastrin, however, increased ODC activity more than 40-fold. In contrast, there was no synergistic induction of ODC activity when gastrin and EGF were added together. Increased ODC activity in cells treated with asparagine and EGF or gastrin was associated with an increase in ODC mRNA and protein levels. The rate of transcription of the ODC gene was significantly increased by exposure to EGF or gastrin. Asparagine alone had little or no effect on the rate of transcription of the ODC gene. When given together with EGF or gastrin, asparagine also had no additional effect on the transcription rate of the ODC gene. The half-life of mRNA for ODC in unstimulated IEC-6 cells was ∼30 min and increased to more than 2 h in cells exposed to asparagine, although neither gastrin nor EGF prolonged the stability of ODC mRNA. The half-life of mRNA for ODC after combined addition of asparagine and EGF or gastrin was extended to ∼2 h, similar to asparagine alone. Combined addition of asparagine and EGF or gastrin also significantly increased DNA synthesis compared with cells exposed to each of the three agents alone. In conclusion, 1) simultaneous addition of asparagine and EGF or gastrin increases ODC activity in a synergistic manner and 2) asparagine increases ODC mRNA levels through completely distinct mechanisms from EGF or gastrin. EGF or gastrin specifically stimulates transcription of the ODC gene, whereas asparagine affects a posttranscriptional process.

Abstract P007: Cardiomyocyte Damage-Released Extracellular S100A1 Protein Promotes Regeneration of Infarcted Myocardium by Modulating Cardiac Fibroblast Function

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
David Rohde ◽  
Gang Qiu ◽  
Nicole Herzog ◽  
Hugo A Katus ◽  
Angelika Bierhaus ◽  
...  
Keyword(s):  
Gene Expression ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Gene Expression Pattern ◽  
Left Ventricular ◽  
Apical Region ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Cardiomyocytes

Background: Similar to heart muscle-specific creatinkinase (CK-MB), S100A1 protein is released from damaged human cardiomyocytes in response to myocardial infarction (MI). Since S100A1-knock out (SKO) mice display rapid post-MI onset of adverse myocardial remodeling and accelerated transition to heart failure, we assessed the hypothesis that ischemia-related release of S100A1 protein modulates myocardial regeneration. Methods and Results: After LAD ligation in C57/B6 mice, S100A1 serum levels peaked at 10 µg/ml 8 hours post-MI, precisely mirroring the time course previously observed in MI patients. RT-PCR analyses in post-MI whole heart samples revealed significantly lower I-CAM (−50%) and IL-10 (−75%) mRNA abundance as well as heightened Collagen-1 (+40%) and VEGF (+80%) expression in SKO vs. WT mice (p<0.05, n=6 in each group). Interestingly, injection of an S100A1-neutralizing antibody prior to MI in WT mice mimicked the abnormalities observed in post-ischemic SKO animals. To further elucidate extracellular S100A1 biological activity, cardiomyocytes, cardiac fibroblasts (CF), endothelial and smooth muscle cells were exposed to S100A1 in vitro . A rapid internalization of S100A1 was exclusively found in CF, resulting in a phosphorylation of ERK1/2, JNK, and p38 with subsequent activation of NF-kappaB as assessed by Western Blot (WB) and EMSA. RT-PCR and WB analyses revealed significant alterations in CF gene expression in response to S100A1, including an increase in I-CAM (3,5-fold) and IL-10 (20-fold) mRNA levels and diminished Col-1 (−80%) expression. Similar effects were observed after direct injection of S100A1 protein into the left ventricular apical region of WT mice in vivo (S100A1- vs. PBS-injection, n=6). In SKO mice, intraperitoneal application of S100A1 prior to MI largely normalized the adverse gene expression pattern towards WT animals. Conclusions: Our study provides first evidence for cardiomyocyte damage-released S100A1 to act as an endogenous mediator of post-MI inflammation and tissue repair. Considering today's unability to manipulate these molecular mechanisms, extracellular S100A1 might represent a promising target for future therapies of MI.

scholarly journals Enhanced AP-1 and NF-κB activities and stability of interleukin 8 (IL-8) transcripts are implicated in IL-8 mRNA superinduction in lung epithelial H292 cells

1998 ◽  
Vol 330 (1) ◽  
pp. 429-435 ◽  
Author(s):  
Thierry ROGER ◽  
A. Theo OUT ◽  
Naofumi MUKAIDA ◽  
Kouji MATSUSHIMA ◽  
M. Henk JANSEN ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Dna Binding ◽  
Mrna Expression ◽  
Gene Transcription ◽  
Molecular Mechanisms ◽  
Interleukin 8 ◽  
Mrna Levels ◽  
Tnf Α ◽  
Lung Epithelial ◽  
Inhibitor Of Protein Synthesis

Inhibition of protein synthesis may result in superinduction of short-lived transcripts and has been attributed variably to stabilization of transcripts and/or increased gene transcription. Little is known about the kinetics of these processes and relevant transcriptional elements have not been identified. In this study, we describe superinduction of interleukin 8 (IL-8) mRNA, an important inflammatory mediator, in lung epithelial-like H292 cells and identify the underlying molecular mechanisms and their kinetics. Cycloheximide (CHI, 10 μg/ml), an inhibitor of protein synthesis, maximally increased IL-8 mRNA levels 30-fold in H292 cells. Tumour necrosis factor α (TNF-α), which induced IL-8 mRNA 3-fold, synergized with CHI causing a 150-fold increase at 6 h. CHI early on increased the stability of IL-8 mRNA (from 40 min in cells cultured with medium to more than 4 h with CHI). CHI also increased transcription as shown by transfection with IL-8 promoter constructs. Truncated and mutated constructs identified NF-κB and AP-1 binding sites as primary cis-acting elements in IL-8 gene transcription and IL-8 mRNA superinduction. Electrophoretic mobility shift assays indicated that CHI increased NF-κB and prolonged AP-1 DNA-binding activities and that the synergism of TNF-α and CHI on IL-8 mRNA expression was paralleled by a further increase of AP-1 DNA-binding activity. This synergism was still noticed when 4 h elapsed between the addition of CHI and that of TNF-α. Taken together, our results indicate that CHI interferes with both post-transcriptional and transcriptional repressive mechanisms of IL-8 mRNA expression.

Induction of GLUT1 mRNA in response to inhibition of oxidative phosphorylation

1993 ◽  
Vol 265 (5) ◽  
pp. C1224-C1229 ◽  
Author(s):  
M. Shetty ◽  
N. Ismail-Beigi ◽  
J. N. Loeb ◽  
F. Ismail-Beigi
Keyword(s):  
Oxidative Phosphorylation ◽  
Gene Transcription ◽  
Half Life ◽  
Actinomycin D ◽  
Late Phase ◽  
Mrna Induction ◽  
Mrna Content ◽  
Glut1 Mrna ◽  
Glut1 Gene ◽  
In Cells

In previous studies, we have shown that inhibition of oxidative phosphorylation in Clone 9 cells (a nontransformed rat liver cell line) by 5 mM azide results in a marked biphasic stimulation of glucose transport that is mediated by GLUT1 [M. Shetty, J. N. Loeb, and F. Ismail-Beige. Am.J. Physiol. 262 (Cell Physiol. 31): C527-C532, 1992]. The late phase of the response (at 8-24 h) is associated with a doubling of cell GLUT1 content and an 8- to 10-fold increment in GLUT1 mRNA abundance. To investigate the mechanisms mediating GLUT1 mRNA induction, we have examined the effect of incubation in the presence of azide on GLUT1 gene transcription. In nuclear run-on assays, the rate of GLUT1 gene transcription was increased 2.5 +/- 0.3-fold in nuclei from cells exposed to azide for 4 h. Additionally, GLUT1 mRNA turnover was decreased in cells treated with azide: upon inhibition of RNA synthesis by actinomycin D, GLUT1 mRNA content decreased with half-lives of 2.3 +/- 0.3 and 8.0 +/- 0.5 h in control cells and cells treated with azide for 4 h, respectively. GLUT1 mRNA half-life was most prolonged (> 12 h) when azide was added subsequent to the addition of actinomycin D, and the half-life continued to be prolonged (6.5 +/- 0.5 h) in cells exposed to azide for 16 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of all trans-retinoic acid and glucocorticoid regulation of surfactant protein mRNA

1998 ◽  
Vol 274 (4) ◽  
pp. L560-L566 ◽  
Author(s):  
Thomas N. George ◽  
Olga L. Miakotina ◽  
Kelli L. Goss ◽  
Jeanne M. Snyder
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Gene Transcription ◽  
Mrna Stability ◽  
Half Life ◽  
Normal Function ◽  
Mrna Levels ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Mrna Half Life

The surfactant proteins (SPs) are required for the normal function of pulmonary surfactant, a lipoprotein substance that prevents alveolar collapse at end expiration. We characterized the effects of cortisol and all trans-retinoic acid (RA) on SP-A and SP-B gene expression in H441 cells, a human pulmonary adenocarcinoma cell line. Cortisol, at 10−6M, caused a significant inhibition of SP-A mRNA to levels that were 60–70% of controls and a five- to sixfold increase in the levels of SP-B mRNA. RA alone (10−6M) had no effect on SP-A mRNA levels and modestly reduced the inhibitory effect of cortisol. RA alone and the combination of cortisol and RA both significantly increased SP-B mRNA levels. RA had no effect on the rate of SP-A gene transcription or on SP-A mRNA stability. Cortisol alone and the combination of cortisol and RA significantly inhibited the rate of SP-A gene transcription but had no effect on SP-A mRNA half-life. RA at 10−6 M had no effect on the rate of SP-B gene transcription but prolonged SP-B mRNA half-life. Cortisol alone and the combination of cortisol and RA caused a significant increase in the rate of SP-B gene transcription and also caused a significant increase in SP-B mRNA stability. We conclude that RA has no effect on SP-A gene expression and increases SP-B mRNA levels by an effect on SP-B mRNA stability and not on the rate of SP-B gene transcription. In addition, the effects of the combination of RA and cortisol were generally similar to those of cortisol alone.

scholarly journals Differential regulation of glutathione peroxidase by selenomethionine and hyperoxia in endothelial cells

1995 ◽  
Vol 306 (2) ◽  
pp. 581-587 ◽  
Author(s):  
L Jornot ◽  
A F Junod
Keyword(s):  
Endothelial Cells ◽  
Glutathione Peroxidase ◽  
Gene Transcription ◽  
Umbilical Vein ◽  
Specific Inhibitor ◽  
Mrna Levels ◽  
Normal Medium ◽  
Initiation Step ◽  
Umbilical Vein Endothelial Cells ◽  
In Cells

We have studied the effect of selenomethionine (SeMet) and hyperoxia on the expression of glutathione peroxidase (GP) in human umbilical vein endothelial cells. Incubation of HUVEC with 1 x 10(-6) M SeMet for 24 h and 48 h caused a 65% and 86% increase in GP activity respectively. The same treatment did not result in significant changes in GP gene transcription and mRNA levels. Pactamycin, a specific inhibitor of the initiation step of translation, prevented the rise in GP activity induced by SeMet and caused an increase in GP mRNA in both cells grown in normal and SeMet-supplemented medium. Interestingly, SeMet supplementation stimulated the recruitment of GP mRNA from an untranslatable pool on to polyribosomes, so that the concentration of GP mRNA in polyribosomal translatable pools was 50% higher in cells grown in SeMet-supplemented medium than in cells grown in normal medium. On the other hand, cells exposed to 95% O2 for 3 days in normal medium showed a 60%, 394% and 81% increase in GP gene transcription rate, mRNA levels and activity respectively. Hyperoxia also stabilized GP mRNA. Hyperoxic cells grown in SeMet-supplemented medium did not show any change in GP gene transcription and mRNA levels, but expressed an 81% and 100% increase in GP activity and amount of GP mRNA associated with polyribosomes respectively, when compared with hyperoxic cells maintained in normal medium. Thus, GP appeared to be regulated post-transcriptionally, most probably co-translationally, in response to selenium availability, and transcriptionally and post-transcriptionally in response to oxygen.

scholarly journals Anemopsis californica Attenuates Photoaging by Regulating MAPK, NRF2, and NFATc1 Signaling Pathways

Antioxidants ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1882
Author(s):  
Quynh T. N. Nguyen ◽  
Minzhe Fang ◽  
Nhung Quynh Do ◽  
Jeehaeng Jeong ◽  
Sarang Oh ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Chronic Inflammation ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Biological Effects ◽  
Dermal Fibroblasts ◽  
Related Factor ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Anemopsis Californica

Long-term exposure of the skin to solar radiation causes chronic inflammation and oxidative stress, which accelerates collagen degradation. This contributes to the formation of wrinkles and dark spots, skin fragility, and even skin cancer. In this study, Anemopsis californica (AC), a herb from North America that is well known for treating microorganism infection and promoting wound healing, was investigated for its photoprotective effects. The biological effects of AC were studied on two in vitro models, namely, lipopolysaccharide (LPS)-induced macrophages and ultraviolet B (UVB)-irradiated dermal fibroblasts, to characterize its underlying molecular mechanisms. The results showed that AC decreased the mRNA levels of inflammatory mediators in sensitized macrophages, including cytokines, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX-2). Moreover, AC alleviated UVB-induced photoaging in dermal fibroblasts by restoring procollagen synthesis. This resulted from the regulation of excessive reactive oxygen species (ROS) by AC, which was mediated by the activation of the antioxidative system nuclear factor erythroid 2-related factor 2 (NRF2). AC also alleviated oxidative stress and inflammatory responses by inhibiting the phosphorylation of mitogen-activated protein kinase (MAPK) and interfering with the nuclear translocation of the immune regulator nuclear factor of activated T-cells 1 (NFATc1). In conclusion, the protective effects of AC on skin cellular components suggested that it has the potential for use in the development of drugs and cosmetics that protect the skin from UVB-induced chronic inflammation and aging.

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