Expression of the macrophage colony stimulating factor CSF-1 and its receptor, the c-fms proto-oncogene, has been observed in macrophages, trophoblast and in a variety of neoplasms of epithelial ...origin including those of the breast. We have reported earlier (Oncogene, 1991, 6: 941-952) that c-fms transcript and protein expression were dramatically increased in several breast carcinoma cell lines by glucocorticoids which are essential humoral regulators of normal mammary epithelial cell differentiation. In this communication, we demonstrate that levels of c-fms transcript and protein increased significantly within the first few hours of glucocorticoid treatment, and that these increases were completely abolished by pretreatment of cells with mifepristone (RU486). We also demonstrate that such early increases in c-fms transcript levels could not be attributed to prolongation of transcript half-life. Both promoters of the c-fms gene were found to exhibit some basal activity in breast carcinoma cell lines and both were stimulated 2-3-fold by glucocorticoids. However the first promoter was shown to be responsible for more than 95% of the observed c-fms transcription. Sequence upstream of both promoters was found to contain potential 'glucocorticoid response elements' (GREs), and in each case, elimination of the GRE closest to the promoter abolished glucocorticoid stimulation. Our observations suggest that one mechanism by which glucocorticoids regulate the proliferation and differentiation of neoplastic mammary epithelial cells is through their regulation of transcription of the gene for the receptor of a ubiquitous cytokine, CSF-1.
To systematically review the evidence on the use of inhaled nitric oxide (iNO) in preterm infants born at or before 34 weeks gestation age who receive respiratory support.
We searched MEDLINE, ...EMBASE, the Cochrane Central Register of Controlled Studies (CENTRAL) and PsycInfo in June 2010. We also searched the proceedings of the 2009 and 2010 Pediatric Academic Societies Meeting and ClinicalTrials.gov. We identified additional studies from reference lists of eligible articles and relevant reviews, as well as from technical experts.
Questions were developed in collaboration with technical experts, including the chair of the upcoming National Institutes of Health Office of Medical Applications of Research Consensus Development Conference. We limited our review to randomized controlled trials (RCTs) for the question of survival or occurrence of bronchopulmonary dysplasia (BPD) and for the question on short-term risks. All study designs were considered for long-term pulmonary or neurodevelopmental outcomes, and for questions about whether outcomes varied by subpopulation or by intervention characteristics. Two investigators independently screened search results, and abstracted data from eligible articles.
We identified a total of 14 RCTs, reported in 23 articles, and eight observational studies. Mortality rates in the NICU did not differ for infants treated with iNO versus those not treated with iNO (RR 0.97 (95% CI 0.82, 1.15)). BPD at 36 weeks for iNO and control groups also did not differ (RR 0.93 (0.86, 1.003) for survivors). A small difference was found between iNO and control infants in the composite outcome of death or BPD (RR 0.93 (0.87, 0.99)). There was inconsistent evidence about the risk of brain injury from individual RCTs, but meta-analyses showed no difference between iNO and control groups. We found no evidence of differences in other short term risks. There was no evidence to suggest a difference in the incidence of cerebral palsy (RR 1.36 (0.88, 2.10)), neurodevelopmental impairment (RR 0.91 (0.77, 1.12)), or cognitive impairment (RR 0.72 (0.35, 1.45)). Evidence was limited on whether the effect of iNO varies by subpopulation or by characteristics of the therapy (timing, dose and duration, mode of delivery, or concurrent therapies).
There was a seven percent reduction in the risk of the composite outcome of death or BPD at 36 weeks PMA for infants treated with iNO compared to controls, but no reduction in death or BPD alone. Further studies are needed to explore particular subgroups of infants and to assess long term outcomes including function in childhood. There is currently no evidence to support the use of iNO in preterm infants with respiratory failure outside the context of rigorously conducted randomized clinical trials.
Late in gestation lung epithelium changes from net chloride and fluid secretion to sodium and fluid absorption. Fluid resorption is required for postnatal gas exchange and occurs by combined action ...of epithelial sodium channels and Na, K-ATPase. We hypothesized that alveolar epithelial Na, K-ATPase increases perinatally. Immunofluorescence (IF) and immunoelectron microscopy (IEM) with a monoclonal anti-alpha subunit antibody demonstrated that Na, K-ATPase was present on the basolateral surfaces of columnar epithelial cells at fetal day (FD) 17 and on type II cells throughout development. However, type I epithelial cells did not have detectable Na,K-ATPase. The steady-state levels of both the alpha 1 isoform and the beta-subunit mRNAs were maximal at FD20-neonatal day (ND) 1, with consistent increases from FD17 level. Na, K-ATPase alpha-subunit protein also increased from FD17 to FD20-22 and then decreased in the early postnatal period. The ouabain-inhibitable sodium pump activity per milligram membrane protein increased 2.6-fold from FD17 to FD22-ND1 (P < 0.05). The quantities of sodium pump mRNA, antigenic protein, and enzyme activity increase in late gestation in accord with a proposed role for Na, K-ATPase in resorption of alveolar sodium and fluid in preparation for birth.
We studied expression of isoforms of Na,K-ATPase in normal and diseased human hearts. Na,K-ATPase α-isoform mRNA in samples from normal human left ventricle (LV) was composed of 62.5% α1, 15% α2 and ...22.5% α3 on average. There was an increase in expression of the α3 isoform in samples from failing hearts, but expression of all three isoforms decreased in pressure-overloaded right ventricle (RV).
The isolation of multiple Na+,K+-ATPase cDNAs from rat brain has led to the discovery of a family of α -isoform genes. Using A1 (α ), A2 (α +), and A3 (α III) Na+,K+-ATPase gene probes, we have ...analyzed the distribution of Na+,K+-ATPase mRNAs in adult and fetal rat tissues by RNA blot and hybridization histochemistry. A1 Na+,K+-ATPase mRNA was found ubiquitously among various tissues, with highest levels in transport epithelial and neural tissues. A2 mRNA was found in adult neural and muscle tissues, and A3 mRNA was found only in neural tissues and fetal heart muscle. Both A1 and A2 mRNAs were less abundant in fetal brain than in adult brain; in contrast, A3 mRNA was abundant at both stages. In situ mapping of brain areas that contain A3 mRNA suggests that this Na+,K+-ATPase isoenzyme is expressed predominantly by neural cells. Analysis of Na+,K+-ATPase proteins generated by cell-free translation of synthetic mRNAs suggests that the A3 protein has properties similar to A2 (α +).
Expression of the Na,K-ATPase alpha and beta subunit genes is influenced by a complex series of regulatory pathways. For example,
unequal amounts of subunit mRNAs are detected in several tissues, ...both at rest and upon mRNA induction, even though equal
quantities of subunit proteins exist. We therefore studied mRNA stability and translational efficiency of wild type, deletion
mutant, and chimeric subunit mRNAs in a cell-free translation system, to examine the possible role of post-transcriptional
events in regulating subunit expression. alpha 1 mRNA translated less efficiently than beta 1 mRNA and competed less efficiently
for rate-limiting translation factors. Deletion of the 5'-untranslated region of alpha 1 mRNA significantly increased its
translation efficiency. Conversely, the alpha 1 5'-untranslated region impaired translation of beta 1 mRNA when attached upstream
of the beta 1 coding sequence. This region is G/C-rich, and has a complex mRNA secondary structure. We propose that differential
translational efficiency may contribute to the equal biosynthesis of subunit proteins in those tissues in which subunit mRNAs
either exist in unequal amounts or are differentially induced.
The c-fms proto-oncogene encodes the receptor for a hematopoietic growth factor, CSF-1. Recently, the importance of c-fms and its ligand CSF-1 in malignancies of non-hematopoietic origin, such as ...breast, ovarian, endometrial, pulmonary, and trophoblastic cancers has been recognized. We have previously shown that glucocorticoids induce a large increase in c-fms mRNA and protein levels in breast carcinoma cell lines. In this report, we investigate the mechanism underlying such c-fms overexpression by dexamethasone. We show that dexamethasone treatment of two breast carcinoma cell lines (BT20-c-fms expressor, and SKBR3-co-expressor of both c-fms and CSF-1) does not increase the rate of c-fms gene transcription, suggesting a post-transcriptional mechanism of regulation of c-fms expression by dexamethasone. The effect of protein synthesis inhibition was studied to help determine whether there was a role for intermediary regulatory proteins in the regulation of c-fms expression. We find that several protein synthesis inhibitors interfere with dexamethasone induction of c-fms transcripts, suggesting the existence of regulatory proteins. These regulatory proteins do not appear to be constitutively expressed, as we show no effect of protein synthesis inhibition on c-fms transcript expression in resting BT20 cells. These findings suggest that the putative regulatory proteins are induced by dexamethasone. Furthermore, the addition of a protein synthesis inhibitor, pactamycin, to dexamethasone-treated BT20 cells results in a decrease in c-fms mRNA stability.
In vitro DNA-amplification technique has been utilized to generate a 430 bp fragment of the Na
+,K
+-ATPase, and a 550 bp fragment of a Ca
2+-ATPase (the sarcoplasmic reticulum-type) of
Drosophila ...melanogaster. The oligonucleotide primers for the DNA-amplification (Polymerase Chain Reaction) had been designed on the basis of amino acid sequence motifs - the phosphorylation site and the ATP-binding site - conserved among members of the ATPase protein family. Using the amplified cDNA-segments as probes, we demonstrated that there is one Na
+,K
+-ATPase and one Ca
2+-ATPase (sarcoplasnaic reticulum-type) gene in the
Drosophila genome. Three different mRNA species are processed from the Na
+,K
+-ATPase gene and one from the Ca
2+-ATPase gene. Developmental control in expression of the Ca
2+-ATPase gene was observed.
The α isoforms of the Na
+,K
+-ATPase (Na
+ pump) are expressed with developmental and tissue heterogeneity in rodents and possess different sensitivity to inhibition by ouabain. We directly ...characterized the ouabain sensitivity of the rat A2 (α2) isoform by transfecting NIH 3T3 cells with rat A2. The treated cells exhibit high affinity (40 nM) ouabain binding with a density of 2 pmol/mg protein.
86Rb
+ flux studies confirm that A2 is Functional in this system and that A2 is inhibited by submicromolar concentrations of ouabain. These findings are consistent with measurements of ouabain affinity in tissues which express the A2 isoform.
During granulocytic differentiation of HL60 cells, immediate reduction of ouabain-sensitive potassium flux is observed within the first 12 hours of addition of dimethyl sulfoxide (DMSO). We show that ...gene expression of the alpha 3 isoform of Na+,K(+)-ATPase, which encodes an ouabain-inhibitable Na+,K(+)-ATPase activity, significantly declines during the first 24 hours of granulocytic differentiation by DMSO of HL60 cells. The more common alpha 1 isoform decreases, but more gradually over 72 hours of DMSO induction. Loss of alpha 3 and alpha 1 messenger RNA (mRNA) are due to changes in mRNA decay; their transcription is not altered. alpha 3 mRNA half-life is 3 hours in HL60 cells; upon induction by 16 hours of DMSO, it decreases to approximately 2 hours. alpha 1 transcripts are less sensitive to DMSO induction, with their half-life being 3.5 hours in HL60 cells; upon induction, their half-life decreases to 3 hours. Experiments measuring protein stability confirm that alpha 3 protein is more labile than alpha 1. In uninduced HL60 cells, alpha 3 membrane protein comprises 30% of the total alpha isoforms, and is less stable than alpha 1, with a protein half-life of only 9 hours. Upon DMSO induction, steady-state alpha 3 protein decreases markedly within 10 hours, whereas alpha 1 protein remains stable. These results show that posttranscriptional changes during induction play a major role in the differential regulation of alpha 1 and alpha 3 isoforms of Na+,K(+)-ATPase; regulation of the latter may be important for early granulocytic differentiation, or for one of the differentiated functions of mature granulocytes.
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