Translational Regulation of Prostaglandin Endoperoxide H Synthase-1 mRNA in Megakaryocytic MEG-01 Cells

Prostaglandin endoperoxide H synthase-1 (PGHS-1) is an abundant enzyme in platelets, where it plays a key role in the cascade of prostanoid formation. In platelets, the primary site of PGHS-1 synthesis is in precursor megakaryocytic cells. We have previously shown that in megakaryocytic MEG-01 cells, TPA induces an increase of PGHS-1 mRNA within a few hours, whereas protein increase occurs after several days of treatment. We now report that the delayed increase in PGHS-1 protein is caused by translational regulation. De novo PGHS-1 synthesis, measured using [35S]methionine pulse labeling followed by immunoprecipitation, was detected at day 4 after TPA treatment but not at day 1. To identify a potential element of PGHS-1 mRNA controlling translation, we compared the 3′-untranslated region from different species and identified a 20-nt segment perfectly conserved. The 20-nt segment was used as a probe in RNA gel mobility-shift assays using MEG-01 extracts from control cells or from TPA-treated cells. Four complexes were formed with extracts from control cells or cells treated with TPA for 1 day but were not observed with extracts from cells treated for 4 days. Of the 4 complexes, one was sequence-specific and binding involved uridylate residues and interactions with a 45-kDa protein and a protein doublet of 116 kDa. Binding of this 45/116-kDa complex to the 20-nt conserved cis element most likely regulates negatively PGHS-1 protein accumulation. We have provided evidence that the PGHS-1 gene is regulated at the translational level. Prostaglandin endoperoxide H synthase-1 (PGHS-1) is an abundant enzyme in platelets, where it plays a key role in the cascade of prostanoid formation. In platelets, the primary site of PGHS-1 synthesis is in precursor megakaryocytic cells. We have previously shown that in megakaryocytic MEG-01 cells, TPA induces an increase of PGHS-1 mRNA within a few hours, whereas protein increase occurs after several days of treatment. We now report that the delayed increase in PGHS-1 protein is caused by translational regulation. De novo PGHS-1 synthesis, measured using [35S]methionine pulse labeling followed by immunoprecipitation, was detected at day 4 after TPA treatment but not at day 1. To identify a potential element of PGHS-1 mRNA controlling translation, we compared the 3′-untranslated region from different species and identified a 20-nt segment perfectly conserved. The 20-nt segment was used as a probe in RNA gel mobility-shift assays using MEG-01 extracts from control cells or from TPA-treated cells. Four complexes were formed with extracts from control cells or cells treated with TPA for 1 day but were not observed with extracts from cells treated for 4 days. Of the 4 complexes, one was sequence-specific and binding involved uridylate residues and interactions with a 45-kDa protein and a protein doublet of 116 kDa. Binding of this 45/116-kDa complex to the 20-nt conserved cis element most likely regulates negatively PGHS-1 protein accumulation. We have provided evidence that the PGHS-1 gene is regulated at the translational level. The formation of prostanoids results from a cascade of enzymatic reactions in which the enzyme prostaglandin endoperoxide H synthase (PGHS) 1The abbreviations used are: PGHS, prostaglandin endoperoxide H synthase; TPA, 12-O-tetradecanoylphorbol-13-acetate; MEG-01, human megakaryocytic cell line, UTR, untranslated region; Tx, thromboxane; TLC, thin layer chromatography; EMSA, electrophoretic mobility shift assay; nt, nucleotide. plays a central role (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Google Scholar). Initially, the first substrate, arachidonic acid, is released from membrane phospholipids by phospholipases. The enzyme PGHS, also known as cyclooxygenase, catalyzes the bis oxygenation of arachidonate to generate prostaglandin H2. Finally, prostaglandin H2 serves as a common substrate for the various isomerases which catalyze the final step in the formation of prostaglandins and thromboxane A2 (TxA2). Much attention has been devoted to the step catalyzed by PGHS since it is the target of aspirin and other nonsteroidal anti-inflammatory drugs (2Laneuville O. Breuer D.K. DeWitt D.L. Hla T. Funk C.D. Smith W.L. J. Pharmacol. Exp. Ther. 1994; 271: 927-934Google Scholar). Two distinct PGHS enzymes are known, PGHS-1 and PGHS-2, and both catalyze the formation of prostaglandin H2from arachidonic acid (3Laneuville O. Breuer D.K. Xu N. Huang Z.H. Gage D.A. Watson J.T. Lagarde M. DeWitt D.L. Smith W.L. J. Biol. Chem. 1995; 270: 19330-19336Google Scholar). The two PGHS have different profiles of expression; PGHS-1 is present in almost all tissues, and stimulation with hormones or mitogenic agents does not affect its basal levels significantly (4Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Google Scholar). In contrast, PGHS-2 enzyme becomes detectable in certain contexts such as inflammation, cancer, or on addition of mitogenic agents (5Herschman H.R. Biochim. Biophys. Acta. 1996; 1299: 125-140Google Scholar). Although we have a great understanding of the regulatory mechanisms leading to the induction of PGHS-2 gene, the response elements and factors regulating the expression of PGHS-1gene are essentially unknown. PGHS-1 gene has a TATA-less promoter, is CG rich, and contains multiple potential start sites for transcription (6Xu X.M. Tang J.L. Chen X. Wang L.H. Wu K.K. J. Biol. Chem. 1997; 272: 6943-6950Google Scholar). A promoter study, using human umbilical vein endothelial cells, has shown that the 916-nucleotides sequence located upstream from the transcriptional start site had very low promoter activity and a modest increase of 1.8-fold was reported after TPA treatment (6Xu X.M. Tang J.L. Chen X. Wang L.H. Wu K.K. J. Biol. Chem. 1997; 272: 6943-6950Google Scholar). A few studies have shown variations in PGHS-1 mRNA and protein levels, notably, during the development of ovine lung (7Brannon T.S. North A.J. Wells L.B. Shaul P.W. J. Clin. Invest. 1994; 93: 2230-2235Google Scholar), as well as during TPA-induced differentiation of monocytes (THP-1) cells to a macrophage phenotype (8Smith C.J. Morrow J.D. Roberts L.J. Marnett L.J. Adv. Exp. Med. Biol. 1997; 400: 99-106Google Scholar). Thus far, mechanisms regulating the basal expression ofPGHS-1 gene as well as its induction during development and differentiation have not been described. To study the regulation of expression of the PGHS-1 gene, we have chosen the megakaryocytic cell line MEG-01 induced to differentiate into platelet-like structures on addition of TPA (9Ogura M. Morishima Y. Okumura M. Hotta T. Takamoto S. Ohno R. Hirabayashi N. Nagura H. Saito H. Blood. 1988; 72: 49-60Google Scholar). Using this model, we have monitored the levels of PGHS-1 protein and mRNA during MEG-01 differentiation; the highest levels of PGHS-1 protein were measured in the most differentiated cells, the enucleated platelet-like population, whereas PGHS-1 mRNA levels were greatest in the nucleated adherent population (10Mroske C. Plant M.H. Franks D.J. Laneuville O. Exp. Hematol. 2000; 28: 411-421Google Scholar). We also performed a time-course study and noted that only mRNA levels were increased at day 1 after addition of TPA whereas both mRNA and protein increased at day 4 after stimulation (11Plant M.H. Laneuville O. Biochem. J. 1999; 344: 677-685Google Scholar). The delay in PGHS-1 enzyme accumulation we observed is not unique to MEG-01 cells. Similar to our observation, the levels of PGHS-1 mRNA and protein were maximal at 24 h and 48 h, respectively, after treatment of human astrocyte cells with histone deacetylase inhibitors (12Taniura S. Kamitani H. Watanabe T. Eling T.E. J. Biol. Chem. 2002; 277: 16823-16830Google Scholar). PGHS-1 was also up-regulated by retinoic acid during neuronal differentiation in neuroblastoma cell lines, and PGHS-1 protein increase occurred 1 day after mRNA increase (13Schneider N. Lanz S. Ramer R. Schaefer D. Goppelt-Struebe M. J. Neurochem. 2001; 77: 416-424Google Scholar). These reports of a delayed accumulation of PGHS-1 enzyme relative to the increase in mRNA levels suggest that PGHS-1 protein synthesis could be under the control of a mechanism yet to be defined. In the current study, we report that although the steady-state levels of PGHS-1 mRNA are comparable at days 1 and 4, synthesis of PGHS-1 protein occurred at day 4 only. To elucidate the mechanism involved in the delay of PGHS-1 protein synthesis, we have identified a conserved 20-nt segment in the 3′UTR and have used it in RNA gel shift assays. We provide evidences for specific protein interactions with the 20-nt conserved segment of the PGHS-1 3′UTR in control or in 1-day-TPA-treated cells. The sequence-specific interaction was not observed using extracts from cells treated for 4 days that contain high levels of PGHS-1 protein. Our experiments indicate the presence of RNA-binding proteins (complex C) that bind to the 20-nt conservedcis element of the 3′UTR of PGHS-1 transcript, likely silencing its translation early after TPA stimulation. MEG-01 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum without antibiotics as previously published by our laboratory (11Plant M.H. Laneuville O. Biochem. J. 1999; 344: 677-685Google Scholar). TPA was added to each culture at a final concentration of 16 nm. Control cells were treated with the same (CH3)2SO concentration that TPA-treated cells received (0.0001% v/v). Cells were incubated (37 °C, air/CO2 (19:1)) for 1 or 4 days and were harvested for RNA or protein analysis, enzymatic activity, metabolic labeling, or gel mobility-shift assays. Total RNA was extracted from MEG-01 cells using Trizol reagent (Invitrogen), mainly as we have described previously (11Plant M.H. Laneuville O. Biochem. J. 1999; 344: 677-685Google Scholar). For Northern blot analysis, after gel separation and transfer, nylon membranes were prehybridized in 5× SSC, 1× Denhardt's solution, 50% formamide, 1% SDS, 10% dextran sulfate, 20 mm Tris-Cl for 30 min. at 42 °C with 30 μg/ml of herring sperm DNA. Hybridization was done at 42 °C overnight using random-primed 32P-labeled PGHS-1 cDNA corresponding to the entire open reading frame, as a probe. The membranes were washed twice in 2× SSC/0.05% SDS at room temperature and then once in 0.1× SSC/0.1% SDS at 65 °C for 30 min. The membranes were then exposed on phosphorscreen to visualize mRNA. The 32P-labeled human β-actin cDNA was used as control for the quantity of RNA loaded in each lane. For analysis of ribosomal RNA, total RNA was isolated from 1 × 107MEG-01 cells, resolved on 1% agarose gels, and stained with ethidium bromide. Protein isolation, quantification, and Western blotting were done as described previously (11Plant M.H. Laneuville O. Biochem. J. 1999; 344: 677-685Google Scholar). Samples (10 μg of total protein) were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. The primary anti-PGHS-1 antibody is directed against residues Leu272-Gln283 of hPGHS-1 (gift of Dr. W. L. Smith, Michigan State University), and detected with anti-rabbit IgG linked to horseradish peroxidase (Promega). Protein bands were visualized using the Roche Molecular Biochemicals Chemiluminescence Blotting Substrate detection system and on and The membranes were incubated with an antibody in For the after membranes were incubated with as a control for the was detected with an IgG horseradish peroxidase (Promega). PGHS-1 from was used as a from Dr. W. L. synthesis by MEG-01 cells was measured at 1 day and 4 days after TPA treatment. Cells were from culture by and in of RPMI 1640 medium of The substrate, acid concentration of 42 was and the cell was incubated at °C for 30 min. To the the cells were for at and the medium was from the medium were by 1 of The the prostanoid mainly and arachidonic acid was with 1 of and extracted with of acid The was to under a of and in of the acid, with Samples were to a gel The were by and detected by to for was identified by with an and by MEG-01 cells were cultured as described and treated or not with TPA for 1 or 4 days. were added h at the and 20 and were in (CH3)2SO at mm and 20 respectively, and was in at MEG-01 cells × were treated with TPA for 1 or 4 days. The cells were by at washed in and in 4 of The cells were incubated at °C to the of and by for 30 with [35S]methionine To the cells were in a of 20 mm mm 1% 10% and incubated at 4 °C for 1 immunoprecipitation, an of total of the were and on a PGHS-1 was from by using PGHS-1 IgG and protein in the were resolved by were and to PGHS-1 was in and stained with to was previously and and isolated from agarose The were to to each other 65 °C for and on and to be into the and The the conserved 20-nt the and to the of the the sequence is the site used to identify The same was used to generate the and A and A for probe The total of each probe by this was and were from the corresponding with using RNA in the presence of 20 and mm each of and and mm for h at The were by and with after of with The were on and used in RNA mobility-shift assays after at or at 1 of probe binding on specific in the PGHS-1 20-nt segment the specific binding of the probe and of to used in indicate different into the conserved 20-nt sequence and into the For each formation of complex is reported on the of experiments probe or probe was incubated in presence of control MEG-01 and shown by the complex formation was observed using or whereas complex formation was with probe or The cells were incubated for 1 or 4 days with or without TPA, harvested by and washed twice in and extracts were obtained as described by J.D. Scholar). Protein were with the of a protein to the Binding reactions were with μg of MEG-01 protein and of 32P-labeled transcript, and were incubated for at room temperature in 1× binding 1 mm 1 30 in a total of 20 to the formation of was added to different final and incubated for at room Samples were then on and for in a at a of from the to on in These binding reactions were by In extracts were for at room temperature with different or such as SDS, or addition of the For complex analysis, reactions to as described were resolved on from binding reactions was and and proteins were in mm SDS and at 4 °C for 16 h with was on and to a of and complex protein were resolved on in were visualized with protein gel were then exposed to for detection of proteins linked to the RNA probe. The PGHS-1 PGHS-1 and PGHS-1 activity in TPA-treated MEG-01 cells was The steady-state of PGHS-1 mRNA was measured by Northern blot analysis and was by to β-actin mRNA In with results we have increase in mRNA was detected at at 1 day after TPA treatment and high for at 4 days (11Plant M.H. Laneuville O. Biochem. J. 1999; 344: 677-685Google Scholar). The steady-state of PGHS-1 protein was measured by Western blot analysis 1 PGHS-1 enzyme increased compared with PGHS-1 was detectable PGHS-1 protein after 1 day of TPA stimulation and a after 4 days. a delay in PGHS-1 protein accumulation occurred in TPA-treated MEG-01 cells. The activity of the PGHS-1 enzyme was measured by the cells with the substrate arachidonic acid and the formation of the of The formation of increased to the levels of PGHS-1 low at 1 day after TPA stimulation and high at 4 days 1 that in PGHS-1 protein levels measured by Western blot are not an The that thromboxane synthase protein activity the formation of 1 C) was on the of a study low formation of high levels of thromboxane synthase protein in MEG-01 cells N. Wang L.H. Wu K.K. Biochim. Biophys. Acta. 1995; Scholar). this study reports very in thromboxane synthase levels after TPA stimulation. The low levels of PGHS-1 protein at day 1 after TPA treatment a high of is with a low of protein synthesis or a high of protein In to the of PGHS-1 protein cells were incubated for h with one of the system 20 or The steady-state levels of PGHS-1 protein in presence or of a were in control cells and at day 1 after TPA treatment but high at day 4 a control for known to be by the was In to the levels increased is added to the cells S. C. S. Scholar). PGHS-1 protein was also not detected in 1-day-TPA-treated cells to other drugs such as the or the a high of as an for the of PGHS-1 protein after 1 day of TPA treatment not To the low of synthesis of PGHS-1 protein at 1 day after TPA novo synthesis was measured by with PGHS-1 levels in the were by with PGHS-1 and The of cells treated with TPA for 4 days but essentially PGHS-1 protein was detected after treatment of the cells for 1 day These results suggest that the low of PGHS-1 enzyme at day 1 after TPA treatment is to a of PGHS-1 protein To is an increase in protein synthesis at 4 days after TPA we also proteins the PGHS-1 with the same of cells × at 1 and 4 days after TPA we observed of [35S]methionine cells are treated for 4 days compared with 1 day was also in the TPA a in the protein synthesis and a in ribosomal after 4 days of TPA the synthesis and steady-state levels of PGHS-1 protein were The of regulated is a site of regulation. In most of translational a RNA-binding protein to a element of the for a potential site for we the PGHS-1 from different species and identified a 20-nt sequence perfectly conserved. For the human PGHS-1 transcript, the 20-nt sequence is located at the after the 4 proteins were detected by an RNA electrophoretic mobility-shift by using the PGHS-1 20-nt conserved sequence as a probe. In the presence of of the probe was by extracts from control MEG-01 cells or cells treated with TPA for 1 complexes, and were formed of the complexes was detected in extracts from cells treated with TPA for 4 days. that the formation of complexes with the 20-nt conserved sequence is both and from the cells a the of which increased with the of the TPA To the of protein or of an of protein used in the the extracts were on SDS-PAGE stained with and have shown profiles 4 The of complexes with the MEG-01 with a high of PGHS-1 protein in this as monitored by Western blot These results suggest that the complexes could PGHS-1 proteins regulating PGHS-1 translational activity The sequence of all observed complexes was using an probe that the same site present in the probe and the PGHS-1 20-nt sequence in the shown in only one complex (complex by the was not detected with the probe incubated with extracts from control MEG-01 cells. observed with the TPA was also formed with the probe the formation of complexes and is both and but not sequence of an of the 20-nt probe to the binding reactions the formation of complex in a not The experiments were to study complex which is and and specific to the 20-nt conserved sequence in the PGHS-1 The protein in the complex was by or of the control MEG-01 its addition to the binding of cell extracts with or in presence of SDS, or cell extracts with SDS or all complex formation line 4 to the probe with MEG-01 extracts not complex formation line of the probe with with a cell from control MEG-01 line not to complex that complexes not from binding with or formed complexes and the sequence the were for the binding of To identify the of the conserved sequence that are for complex we performed experiments and binding from control MEG-01 cells were with of or the addition of the probe to the binding shown in the formation of complex was by as low as and by an of or not significantly its formation at concentration The binding activity from MEG-01 extracts sequence with the that 50% of the the 20-nt conserved sequence are of which are we in and used in we 1 of and observed that the formation of complex was but not we the residues in the conserved sequence and binding shown in of the sequence to and the formation of complex C. only the of the residues formation of complex C. the residues of the 20-nt sequence and in the are for the formation of complex C. To the that with the 20-nt conserved region of the PGHS-1 we first on gels, binding reactions to was from the and resolved on a were first stained with to visualize all the were then exposed to a to identify proteins with the RNA probe. The stained gel that complex is of at proteins with from with two bands at and a doublet at 116 and a at were that proteins are likely to bind the 20-nt sequence a first step to PGHS-1 we steady-state levels of PGHS-1 mRNA and protein in a megakaryocytic cell line induced to The levels of PGHS-1 mRNA were maximal at 1 day and for at 4 days after treatment with TPA, PGHS-1 protein was not detected at 1 but levels were high at day We that a delay at the translational step was for the of PGHS-1 protein at day 1 after TPA treatment. To this we novo synthesis of PGHS-1 enzyme with metabolic labeling experiments and PGHS-1 at day but synthesis at day a potential mechanism that the of PGHS-1 translation in control MEG-01 cells or early after TPA we report the binding of and proteins to a 20-nt conserved sequence located in the of the PGHS-1 Our current results suggest that protein extracts from control or 1-day-TPA-treated MEG-01 cells to the 20-nt conserved sequence in the binding to as complexes was in the of PGHS-1 To the the synthesis of PGHS-1 enzyme with the formation of complex T. The conserved sequence in the PGHS-1 to be as a has that it is not in other Similar to previously reported mRNA cis is only one of the 20-nt cis element within the PGHS-1 A has to identify other known elements in the of Y. Chen Biol. Scholar), J. Biol. Chem. 1997; 272: Scholar), S. C. L. Scholar), and Smith Biochem. Biophys. 1996; that translational regulation. These 20-nt have to the PGHS-1 conserved sequence PGHS-1 conserved all contain of uridylate residues for the binding The sequence-specific complex formed (complex C) with the 20-nt conserved sequence proteins of 116 and of that to the RNA probe. on our that the is for protein we that proteins known to bind A the in at are potential S. Annu. Rev. Biochem. Scholar). The of translational regulation for the expression of is only at this The of ribosomal RNA after TPA treatment is with reports of a of ribosomal that differentiation of cells D. J. Biol. Chem. 2001; Scholar). a in ribosomal be to cells to gene expression from proteins involved in and differentiated cells. For the MEG-01 cells, PGHS-1 protein synthesis be a of the levels of the of translational regulation We have reported that PGHS-1 protein levels a increase the entire of differentiation of MEG-01 cells, that from cell to (10Mroske C. Plant M.H. Franks D.J. Laneuville O. Exp. Hematol. 2000; 28: 411-421Google Scholar). is well that one of of PGHS-1 enzyme in the but the mechanisms leading to this are C.D. Funk L.B. J. Scholar). The most likely role of the translational regulation we be to the accumulation of PGHS-1 protein early in In this differentiation of the precursor cells is by the the levels of PGHS-1 mRNA but protein synthesis be delayed differentiation of the cells has the of In a to megakaryocytic differentiation is with the translational silencing of the N. J. 1994; Scholar). mRNA is in the of the but untranslated the of into cells. PGHS-1 enzyme the same substrate as and both enzymes are at high levels in enucleated cells and the cells, In in the of TPA-induced MEG-01 the increase in the levels of PGHS-1 protein could be for by the increase in PGHS-1 synthesis, on [35S]methionine labeling, that translational regulation is a of We have identified a 20-nt conserved region in the of the PGHS-1 as a potential regulatory We report a the of proteins to this cis element and the of PGHS-1 protein We have identified a of residues within the 20-nt conserved sequence that is for binding we have the of the PGHS-1 and to be We for

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