A fluorescence microscopy method for quantifying the detection of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells

In platelets, PGHS-1-dependant formation of thromboxane A₂ is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are anucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01, which can be induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression. Using a specific antibody to PGHS-1 we have developed a technique using immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein expression as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 × 10⁻⁸ M) over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen, CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation between PGHS-1 expression and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein expression, which shows a consistent increase over the entire course of differentiation can be used as an additional and better index by which to monitor megakaryocyte differentiation. Published: December 12, 2001     

Specific protein binding to a conserved region of the ornithine decarboxylase mRNA 5'-untranslated region.

An RNA gel retardation assay was used to identify one or more cellular protein(s) (ornithine decarboxylase mRNA 5'-UTR binding protein (ODCBP)) that bind specifically to a conserved region of the 5'-untranslated region (5'-UTR) of rat ornithine decarboxylase (ODC) mRNA. Ultraviolet light cross-linking demonstrated that this protein has an apparent Mr = 58,000 in mammalian cells. Treatment with the oxidizing agent diamide prevented binding of the ODCBP to ODC mRNA; addition of beta-mercaptoethanol reversed this inhibition and permitted mRNA.ODCBP complex formation. Cytoplasmic extracts from a variety of animal cells and tissues demonstrated similar binding activities; however, there was marked tissue-specific expression of the protein in the rat, with brain, heart, lung, and testis containing large amounts, and kidney, spleen, and skeletal muscle expressing negligible amounts. Binding was completely prevented by several mutations within a highly conserved heptanucleotide region (CCAU/ACUC) that was within 61 bases of the initiation codon in ODC mRNAs from mammals, Xenopus, and Caenorhabditis elegans; mutations 5' and 3' of the conserved heptanucleotide domain had no effect on binding activity. Binding was not affected by manipulation of cellular polyamine levels or by treatment of cells with agents that stimulate ODC biosynthesis. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 5'-UTR of ODC mRNA from many animal species; functional consequences of this binding remain to be determined.     

Growth factor-mediated stabilization of amyloid precursor protein mRNA is mediated by a conserved 29-nucleotide sequence in the 3'-untranslated region

Using a cell-free translation system, we previously demonstrated that the turnover and translation of amyloid precursor protein (APP) mRNA was regulated by a 29-nucleotide instability element, located 200 nucleotides downstream from the stop codon. Here we have examined the regulatory role of this element in primary human capillary endothelial cells under different nutritional conditions. Optimal proliferation required a growth medium (endothelial cell growth medium) supplemented with epidermal, basic fibroblast, insulin-like, and vascular endothelial growth factors. In vitro transcribed mRNAs with the 5'-untranslated region (UTR) and coding region of beta-globin and the entire 3'-UTR of APP 751 were transfected into cells cultured in endothelial cell growth medium. Wild-type globin-APP mRNA containing an intact APP 3'-UTR and mutant globin-APP mRNA containing a mutated 29-nucleotide element decayed with identical half-lives (t 1/2 = 60 min). Removal of all supplemental growth factors from the culture medium significantly accelerated the decay of transfected wild-type mRNA (t 1/2 = 10 min), but caused only a moderate decrease in the half-life of transfected mutant mRNA (t 1/2 = 40 min). We therefore conclude that the 29-nucleotide 3'-UTR element is an mRNA destabilizer whose function can be inhibited by inclusion of the aforementioned mixture of growth factors in the culture medium.     

Different substrate utilization between prostaglandin endoperoxide H synthase-1 and -2 in NIH3T3 fibroblasts

Recent studies suggested that prostaglandin endoperoxide H synthase-1 and prostaglandin endoperoxide H synthase-2 (PGHS-1 and PGHS-2) utilize different pools of arachidonic acid for synthesizing prostanoids. Using cultured murine NIH3T3 fibroblasts, we investigated the mechanism for the different utilization of arachidonic acid between PGHS-1 and -2. Histofluorescence staining for PGHS activity in intact cells demonstrated that quiescent 3T3 cells expressed only PGHS-1 activity and serum-activated 3T3 cells pretreated with aspirin expressed only PGHS-2 activity. Endogenous arachidonic acid released by calcium ionophore A23187 was not converted by PGHS-1 but exclusively converted by PGHS-2. In the cell free system, the kinetics of PGHS-1 were not so much different from those of PGHS-2. However, in intact cells, arachidonic acid at concentrations lower than 2.5 μM was converted by PGHS-2 alone but not by PGHS-1. Our findings indicated that this small amount of arachidonic acid as released by some stimuli is converted exclusively by PGHS-2. Furthermore, treating the PGHS-2-expressing cells with sodium selenite or ebselen, reducing agents of intracellular peroxides, only decreased PGHS-2 activity. We speculate that only PGHS-2 has been activated by intracellular peroxides and subsequently, it can convert the arachidonic acid released endogenously.     

Multiple,conserved iron-responsive elements in the 3'-untranslated region of transferrin receptor mRNA enhance binding of iron regulatory protein 2

Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3'-untranslated region (TfR-3'-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3'-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)(2) binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3'-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.     

Molecular cloning of the mouse S-adenosylmethionine decarboxylase cDNA: specific protein binding to the conserved region of the mRNA 5'-untranslated region.

The nucleotide sequence of a cDNA encoding the proenzyme of mouse S-adenosylmethionine decarboxylase (AdoMetDC) including 257 nucleotides of the 5' untranslated region has been determined. Comparison of the nucleotide sequence of the mouse 5' untranslated region with those of other mammals shows it to be highly conserved. The 52 nucleotides upstream from the translation initiation codon are identical in human, rat, bovine and mouse. The polyamines, spermidine and spermine, have been shown to inhibit AdoMetDC mRNA translation. An RNA gel retardation assay demonstrated that a cytoplasmic extract from mouse brain forms an RNA-protein complex with the completely conserved 5' untranslated sequence and that the complex formation is highly dependent on the presence of spermine. Crosslinking by UV irradiation shows that the complex contains a 39-kDa protein interacting with the 5' untranslated sequence. These data demonstrate spermine-dependent specific protein binding to a highly conserved 5' untranslated region of an mRNA translationally regulated by polyamines.     

Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells.

Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide synthase (NOS II) expression and nitric oxide (NO) production are up-regulated in these diseases as well. However, the apparent link between PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS-2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse colonic epithelial cells, namely YAMC (Apc(+/+)). We found that the induction of PGHS-2 and generation of PGE2 in these cells by IFN-gamma and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGHS-2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR-1 and SNAP caused beta-catenin/LEF-1 DNA complex formation. Super-shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic beta-catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of beta-catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS-2 expression and beta-catenin/LEF-1 DNA complex formation, we studied IMCE (Apc(Min/+)) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their beta-catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher beta-catenin/LEF-1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS-2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble beta-catenin and the formation of nuclear beta-catenin/LEF-1 DNA complex.     

The regulatory element in the 3'-untranslated region of human papillomavirus 16 inhibits expression by binding CUG-binding protein 1

The 3'-untranslated regions (UTRs) of human papillomavirus 16 (HPV16) and bovine papillomavirus 1 (BPV1) contain a negative regulatory element (NRE) that inhibits viral late gene expression. The BPV1 NRE consists of a single 9-nucleotide (nt) U1 small nuclear ribonucleoprotein (snRNP) base pairing site (herein called a U1 binding site) that via U1 snRNP binding leads to inhibition of the late poly(A) site. The 79-nt HPV16 NRE is far more complicated, consisting of 4 overlapping very weak U1 binding sites followed by a poorly understood GU-rich element (GRE). We undertook a molecular dissection of the HPV16 GRE and identify via UV cross-linking, RNA affinity chromatography, and mass spectrometry that is bound by the CUG-binding protein 1 (CUGBP1). Reporter assays coupled with knocking down CUGBP1 levels by small interfering RNA and Dox-regulated shRNA, demonstrate CUGBP1 is inhibitory in vivo. CUGBP1 is the first GRE-binding protein to have RNA interfering knockdown evidence in support of its role in vivo. Several fine-scale GRE mutations that inactivate GRE activity in vivo and GRE binding to CUGBP1 in vitro are identified. The CUGBP1.GRE complex has no activity on its own but specifically synergizes with weak U1 binding sites to inhibit expression in vivo. No synergy is seen if the U1 binding sites are made weaker by a 1-nt down-mutation or made stronger by a 1-nt up-mutation, underscoring that the GRE operates only on weak sites. Interestingly, inhibition occurs at multiple levels, in particular at the level of poly(A) site activity, nuclear-cytoplasmic export, and translation of the mRNA. Implications for understanding the HPV16 life cycle are discussed.     

Differential effects of nitric oxide on the activity of prostaglandin endoperoxide H synthase-1 and -2 in vascular endothelial cells

A number of studies have demonstrated that prostacyclin and nitric oxide (NO) regulate blood pressure, blood flow and platelet aggregation. In this paper, we have examined the possible relationship between NO and prostaglandin endoperoxide H synthase (PGHS)-1 and -2 activities in cultured bovine aortic endothelial cells. In the non-activated condition endothelial cells expressed PGHS-1 activity alone. When these cells were pretreated with aspirin to inactivate their PGHS-1 and then activated by serum and phorbol ester (TPA) for 6 h, the cells expressed PGHS-2 activity alone. The PGHS activity was assessed by the generation of 6-ketoprostaglandin F1alpha (6-ketoPGF1alpha), a stable metabolite of prostacyclin, after the treatment of these cells with arachidonic acid. The simultaneous addition of NOC-7, a NO donor, with arachidonic acid did not affect the production of 6-ketoPGF1alpha in PGHS-1 expressed cells, but attenuated it in PGHS-2-expressed cells. The inhibitory effect of NOC-7 on PGHS-2 activity was dose dependent, and the different effects of NOC-7 on the activities of PGHS isozymes were also observed in other NO donors. To confirm the different effect of NO on PGHS isozymes demonstrated in the cultured endothelial cells, we carried out an ex vivo perfusion assay in aorta isolated from normal and lipopolysaccharide (LPS)-treated rats. In the aortae isolated from normal rats, where dominant expression of PGHS-1 was expected, the NO donor did not affect the PGHS activity, while in aortae isolated from LPS-treated rats, where PGHS-2 was dominantly expressed, the NO donor dramatically inhibited the PGHS activity, suggesting that NO suppressed PGHS-2 activity alone. The inhibitory effect of NO on PGHS-2 activity was not mediated by cyclic GMP (cGMP), since (a) methylene blue, an inhibitor of soluble guanylate cyclase did not abolish the inhibitory effect of the NO donor on PGHS-2 activity, and (b) 8-Br-cGMP, a permeable cGMP analogue, failed to mimic the effect of NO donors. These data suggest that the effect of NO on prostacyclin production in endothelial cells was dependent on the expression rate of PGHS-1 and PGHS-2 in the cells.     

GRSF-1: a poly(A)+ mRNA binding protein which interacts with a conserved G-rich element.

Computer predictions identified similarities to a 14-base G-rich element in numerous mRNAs at a variety of locations. A Northwestern screening strategy was used to obtain a cDNA clone from a HeLa cell library using the G-rich RNA element as a probe. A cellular protein (called GRSF-1), which was encoded by this cDNA, binds RNAs containing the G-rich element. GRSF-1 was distinct from DSEF-1, a nuclear protein we have previously identified that interacts with the G-rich element, based on differences in molecular weight and partial peptide maps, as well as the lack of cross-reactivity with GRSF-1 specific monoclonal antibodies. Using indirect immunofluorescence microscopy, we localized GRSF-1 to the cytoplasm. In vivo UV cross-linking further demonstrated that GRSF-1 was bound to poly(A)+ mRNA in living human cells. Western blot analysis revealed four cytoplasmic proteins which expressed GRSF-1 specific epitopes. GRSF-1 contains three potential RNA recognition motifs and two auxiliary domains. Curiously, the domain organization of GRSF-1 is similar to the RNA binding proteins PUB1, ELAV, HuD, Hel-N1, mcs94-1 and RBP9.     

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.     

The binding of arachidonic acid in the cyclooxygenase active site of mouse prostaglandin endoperoxide synthase-2 (COX-2). A putative L-shaped binding conformation utilizing the top channel region.

The chemical mandates for arachidonic acid conversion to prostaglandin G(2) within the cyclooxygenase (COX) active site predict that the substrate will orient in a kinked or L-shaped conformation. Molecular modeling of arachidonic acid in sheep COX-1 confirms that this L-shaped conformation is possible, with the carboxylate moiety binding to Arg-120 and the omega-end positioned above Ser-530 in a region termed the top channel. Mutations of Gly-533 to valine or leucine in the top channel of mCOX-2 abolished the conversion of arachidonic acid to prostaglandin G(2), presumably because of a steric clash between the omega-end of the substrate and the introduced side chains. A smaller G533A mutant retained partial COX activity. The loss of COX activity with these mutants was not the result of reduced peroxidase activity, because the activity of all mutants was equivalent to the wild-type enzyme and the addition of exogenous peroxide did not restore full COX activity to any of the mutants. However, the Gly-533 mutants were able to oxidize the carbon 18 fatty acid substrates linolenic acid and stearidonic acid, which contain an allylic carbon at the omega-5 position. In contrast, linoleic acid, which is like arachidonic acid in that its most omega-proximal allylic carbon is at the omega-8 position, was not oxidized by the Gly-533 mutants. Finally, the ability of Gly-533 mutants to efficiently process omega-5 allylic substrates suggests that the top channel does not serve as a product exit route indicating that oxygenated substrate diffuses from the cyclooxygenase active site in a membrane proximal direction.     

Cis-acting sequences in the 5'-untranslated region of the ribosomal protein A1 mRNA mediate its translational regulation during early embryogenesis of Drosophila.

The rate of ribosomal (r)-protein synthesis in the early Drosophila embryo is low despite the presence of abundant, maternally supplied r-protein mRNAs. This low rate is due to specific repression of r-protein mRNA translation. In contrast to r-protein mRNAs, most other mRNAs are efficiently translated in the early embryo. Here we report on the identification of cis-acting sequences that mediate translational repression of the r-protein A1 (rpA1) mRNA. Chimeric genes containing sequences from the translationally regulated rpA1 mRNA fused to the constitutively translated alpha-tubulin mRNA were constructed and transformed into the Drosophila germ line. Translation of the corresponding hybrid mRNAs was measured in ovaries and embryos of the transgenic flies. The results indicated that a 89-nucleotide sequence in the untranslated rpA1 mRNA leader is by itself sufficient to confer full translational regulation to a heterologous mRNA.