Sequences of a hairpin structure in the 3'-untranslated region mediate regulation of human pulmonary surfactant protein B mRNA stability

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate expression of surfactant protein B (SP-B). Dexamethasone (DEX, 10(-7) M) increases human SP-B mRNA stability by a mechanism that requires a 126-nt-long segment (the 7.6S region) of the 3'-untranslated region (3'-UTR). The objective of this study was to identify sequences in the 7.6S region that mediate regulation of SP-B mRNA stability. The 7.6S region was found to be sufficient for DEX-mediated stabilization of mRNA. Sequential substitution mutagenesis of the 7.6S region indicates that a 90-nt region is required for DEX-mediated stabilization and maintenance of intrinsic stability. In this region, one 30-nt-long element (002), predicted to form a stem-loop structure, is sufficient for DEX-mediated stabilization of mRNA and intrinsic mRNA stability. Cytosolic proteins specifically bind element 002, and binding activity is unaffected whether proteins are isolated from cells incubated in the absence or presence of DEX. While loop sequences of element 002 have no role in regulation of SP-B mRNA stability, the proximal stem sequences are required for DEX-mediated stabilization and specific binding of proteins. Mutation of the sequences that comprise the proximal or distal arm of the stem negates the destabilizing activity of element 002 on intrinsic SP-B mRNA stability. These results indicate that cytosolic proteins bind a single hairpin structure that mediates intrinsic and hormonal regulation of SP-B mRNA stability via mechanisms that involve sequences of the stems of the hairpin structure.     

Effect of cigarette smoke and dexamethasone on Hsp72 system of alveolar epithelial cells

Smoking is the leading risk factor of chronic obstructive pulmonary disease (COPD) and lung cancer. Corticosteroids are abundantly used in these patients; however, the interaction of smoking and steroid treatment is not fully understood. Heat shock proteins (Hsps) play a central role in the maintenance of cell integrity, apoptosis and cellular steroid action. To better understand cigarette smoke-steroid interaction, we examined the effect of cigarette smoke extract (CSE) and/or dexamethasone (DEX) on changes of intracellular heat shock protein-72 (Hsp72) in lung cells. Alveolar epithelial cells (A549) were exposed to increasing doses (0; 0.1; 1; and 10 μM/μl) of DEX in the medium in the absence(C) and presence of CSE. Apoptosis, necrosis, Hsp72 messenger-ribonucleic acid (mRNA) and protein expression of cells were measured, and the role of Hsp72 on steroid effect examined. CSE reduced the number of viable cells by significantly increasing the number of apoptotic and necrotic cells. DEX dose-dependently decreased the ratio of apoptosis when CSE was administered, without change in necrosis. CSE − DEX co-treatment dose-dependently increased Hsp72 mRNA and protein expression, with the highest level measured in CSE + DEX (10) cells, while significantly lower levels were noted in all respective C groups. Pretreatment with Hsp72 silencing RNA confirmed that increased survival observed following DEX administration in CSE-treated cells was mainly mediated via the Hsp72 system. CSE significantly decreases cell survival by inducing apoptosis and necrosis. DEX significantly increases Hsp72 mRNA and protein expression only in the presence of CSE resulting in increased cellular protection and survival. DEX exerts its cell protective effects by decreasing apoptotic cell death via the Hsp72 system in CSE-treated alveolar epithelial cells.     

Regulation of human pulmonary surfactant protein gene expression by 1alpha,25-dihydroxyvitamin D3

1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has been reported to stimulate lung maturity, alveolar type II cell differentiation, and pulmonary surfactant synthesis in rat lung. We hypothesized that 1,25(OH)(2)D(3) stimulates expression of surfactant protein-A (SP-A), SP-B, and SP-C in human fetal lung and type II cells. We found that immunoreactive vitamin D receptor was detectable in fetal lung tissue and type II cells only when incubated with 1,25(OH)(2)D(3). 1,25(OH)(2)D(3) significantly decreased SP-A mRNA in human fetal lung tissue but did not significantly decrease SP-A protein in the tissue. In type II cells, 1,25(OH)(2)D(3) alone had no significant effect on SP-A mRNA or protein levels but reduced SP-A mRNA and protein in a dose-dependent manner when the cells were incubated with cAMP. SP-A mRNA levels in NCI-H441 cells, a nonciliated bronchiolar epithelial (Clara) cell line, were decreased in a dose-dependent manner in the absence or presence of cAMP. 1,25(OH)(2)D(3) had no significant effect on SP-B mRNA levels in lung tissue but increased SP-B mRNA and protein levels in type II cells incubated in the absence or presence of cAMP. Expression of SP-C mRNA was unaffected by 1,25(OH)(2)D(3) in lung tissue incubated +/- cAMP. These results suggest that regulation of surfactant protein gene expression in human lung and type II cells by 1,25(OH)(2)D(3) is not coordinated; 1,25(OH)(2)D(3) decreases SP-A mRNA and protein levels in both fetal lung tissue and type II cells, increases SP-B mRNA and protein levels only in type II cells, and has no effect on SP-C mRNA levels.     

Inhibition of the production of mediators of inflammation by corticosteroids is a glucocorticoid receptor-mediated process

In order to find an explanation for corticosteroid resistance we assessed whether inhibition by dexamethasone (DEX) of the stimulated production of TNF- proportional, variant, IL-6, PGE(2) and LTB(4) by peripheral blood mononuclear cells (MNC) depends on binding to the glucocorticoid receptor (GR), and whether it is determined by the number or the affinity of the GR of these cells. GR number and affinity of MNC were determined by means of a whole cell DEX binding assay. MNC were incubated with DEX and LPS or A23187 in the absence or presence of RU486, a potent steroid antagonist. DEX caused a concentration dependent inhibition of TNF- proportional, variant, IL-6 and PGE(2) production but had no effect on LTB(4) production. RU486 significantly blocked the effect of DEX, but no correlations were found between the inhibition of mediator release and the K(d) or receptor number.     

Estradiol inhibits glucocorticoid receptor expression and induces glucocorticoid resistance in MCF-7 human breast cancer cells

Our study has shown that treatment of MCF-7 human breast cancer cells with 17-beta estradiol (E(2)) produced significant decreases in glucocorticoid receptor (GR) concentrations and GR mRNA levels. E(2) pre-treatment of MCF-7 cells stably transfected with the GR responsive pMTV-CAT reporter (MCF-7-MTV cells), caused significant attenuation of dexamethasone (DEX)-induced chloramphenicol acetyl transferase (CAT). In MCF-7 cells transiently transfected with [(GRE)(3)-Luc] reporter plasmid, E(2) pre-treatment significantly suppressed DEX-induced luciferase, which was abolished by the estrogen receptor antagonist ICI 182,780. We examined the effect of chronic E(2) treatment as well as E(2) withdrawal on GR function and abundance. MCF-7-MTV cells were treated with vehicle (control) or E(2) for up to 16 days. A third group received E(2) for 5 days followed by E(2) withdrawal from day 6 to 16. Chronic E(2) treatment almost totally abrogated DEX-induced CAT and reduced GR to very low levels. Interestingly, in the group subjected to E(2) withdrawal, neither the DEX response nor GR abundance recovered and reached control values suggesting that the estrogen mediated suppression is long lasting and could not be easily reversed. The E(2) induced resistance to glucocorticoid action may be of potential clinical significance in a number of settings including breast cancer, neuroendocrine response to stress and osteoporosis and could possibly contribute to the differences in glucocorticoid responsiveness among patients.     

Expression and induction of CYP3As in human fetal hepatocytes

CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital. Testosterone 6beta-hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6beta-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5microM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR.     

Differences in nuclear retention characteristics of agonist-activated glucocorticoid receptor may determine specific responses

We studied the glucocorticoid response to the synthetic steroid pregna-1,4-diene-11beta-ol-3,20-dione (DeltaHOP) in several cell types and correlated its biological effect with the ability of the glucocorticoid receptor (GR) to be retained in the nuclear compartment. We observed that the DeltaHOP-transformed GR was diffusely distributed in the nucleus compared to the discrete structures observed for the dexamethasone (DEX)-transformed GR. Despite the fact that the receptor was entirely nuclear upon binding of each steroid and exhibited identical nuclear export rates, a greater amount of DeltaHOP-transformed GR was recovered in the cytoplasmic fraction after hypotonic cell lysis. Furthermore, accelerated nuclear export of GR was evidenced in digitonin-permeabilized cells treated with ATP and molybdate. Inasmuch as limited trypsinization of DEX-GR and DeltaHOP-GR complexes yielded different proteolytic products, we conclude that GR undergoes a differential conformational change upon binding of each ligand. We propose that these conformational differences may consequently lead to changes of stability in the interaction of the GR with chromatin. Therefore, the dynamic exchange of liganded GR with chromatin is likely to have significant consequences for the observed pleiotropic physiological responses triggered by glucocorticoid ligands, not only in different tissues but also in the same cell type.     

Cell type-specific regulation of fetal fibronectin expression in amnion: conservation of glucocorticoid responsiveness in human and nonhuman primates

The appearance of oncofetal fibronectin (FFN) in cervical and vaginal secretions is predictive of human labor. Levels of FFN in amnion increase with the onset of labor in rhesus monkeys. Since glucocorticoid (GC) levels in serum and amniotic fluid increase in association with parturition, we compared GC-mediated regulation of FFN expression in cultures of amnion epithelial cells and fibroblasts isolated from human and baboon amnions. Cells were maintained with and without dexamethasone (DEX), and levels of FFN in the conditioned media were determined by ELISA. We observed that DEX treatment suppressed FFN levels in both human and baboon amnion epithelial cells, whereas it increased FFN levels in amnion fibroblasts. DEX treatment reduced FFN levels in cytotrophoblasts from human placenta and increased FFN levels in placental fibroblasts. Northern blots revealed that DEX reduced levels of fibronectin (FN) mRNA in amnion epithelial cells and cytotrophoblasts, whereas it increased FN mRNA in amnion and placental fibroblasts. We conclude that GC differentially regulates FFN expression in epithelial and mesenchymal cells from amnion and placenta. In addition, this pattern of cell type-specific FFN regulation by GC is conserved in human and nonhuman primates and may be responsible for parturition-dependent changes in FFN expression in gestational tissues.     

Rapid inhibition of c-myc gene expression by a glucocorticoid in the avian oviduct

The glucocorticoid dexamethasone (DEX) causes a rapid, reversible reduction in c-myc mRNA level in the oviducts of estrogen-treated, immature chickens. The c-myc mRNA level begins to decrease by 5 min after injection of 0.5 mg DEX, reaches a minimum of 10% of the control value by 30 min, and returns to 30-40% of the control value by 4 h post injection. This rapid effect of DEX on the c-myc mRNA level occurs in both diethylstilbestrol-stimulated and diethylstilbestrol-withdrawn oviducts. The effect is dose dependent, with reduction of the c-myc mRNA measured with as little as 10 micrograms DEX injection (0.03 micrograms/g BW). The effect of the steroid is gene specific with H2B histone mRNA displaying a significantly reduced response. The effect is also tissue specific with liver displaying an increase of 170% of control values in c-myc mRNA level by 30 min after injection of 0.5 mg DEX. The reduction of avian oviduct c-myc mRNA levels by DEX may play a role in glucocorticoid inhibition of cell proliferation in this tissue. The rapidity of the steroid effects on c-myc expression makes it likely that the steroid-induced reduction of c-myc mRNA levels represents a direct primary action of the steroid-receptor complex on the c-myc gene expression.     

Involvement of β1‐integrin via PIP complex and FAK/paxillin in dexamethasone‐induced human mesenchymal stem cells migration

Although glucocorticoids strongly affect numerous biological processes including cell growth, development, and homeostasis, their effects on migration of human mesenchymal stem cells (hMSCs) are unclear. Therefore, we investigated the role of dexamethasone (DEX) and its related signaling pathways on migration of hMSCs. We found that DEX, at 10^?8 to 10^?6 M, significantly increased migration after a 24 h incubation, and DEX (10^?6 M) increased migration at >12 h. Moreover, DEX (10^?6 M) increased the level of glucocorticoid receptor (GR)‐α mRNA and protein expression, but not GR‐β mRNA. The increases in DEX‐induced migration were inhibited by the GR antagonist mifepristone (10^?7 M). In addition, DEX increased integrin‐linked kinase (ILK) and α‐parvin expression but did not change PINCH‐1/2 expression in lysate. DEX also increased formations of complex with ILK and α‐parvin, and ILK and PINCH‐1/2 as shown by immunoprecipitation, which were all inhibited by mifepristone. DEX‐induced migration was blocked by ILK and α‐parvin small interfering(si)RNAs. In addition, DEX increased focal adhesion kinase (FAK) and paxillin expression, which were attenuated by ILK and α‐parvin siRNAs. DEX‐induced cell migration was inhibited by FAK/paxillin siRNAs. DEX also increased β1‐integrin expression, which was blocked by FAK/paxillin siRNAs. In addition, DEX‐induced cell migration was inhibited by β1‐integrin siRNA. Downregulation of ILK, α‐parvin, FAK/paxillin and β1‐integrin expression by siRNAs decreased DEX‐induced filamentous(F)‐actin organization and migration of hMSCs. In conclusion, DEX partially stimulates hMSC migration by the expression of β1‐integrin through formation of a PINCH‐1/2/ILK/α‐parvin complex (PIP complex), and FAK and paxillin expression. J. Cell. Physiol. 226: 683–692, 2011. © 2010 Wiley‐Liss, Inc.