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• W2086452134 abstract "Hypoxia is a pro-fibrotic stimulus, which is associated with enhanced collagen synthesis, as well as with augmented collagen prolyl 4-hydroxylase (C-P4H) activity. C-P4H activity is controlled mainly by regulated expression of the α C-P4H subunit. In this study we demonstrate that the increased synthesis of C-P4H-α(I) protein in human HT1080 fibroblasts under long term hypoxia (36 h, 1% oxygen) is controlled at the translational level. This is mediated by an interaction of RNA-binding protein nucleolin (∼64 kDa form) at the 5′- and 3′-untranslated regions (UTR) of the mRNA. The 5′/3′-UTR-dependent mechanism elevates the C-P4H-α(I) expression rate 2.3-fold, and participates in a 5.3-fold increased protein level under long term hypoxia. The interaction of nucleolin at the 5′-UTR occurs directly and depends on the existence of an AU-rich element. Statistical evaluation of the ∼64-kDa nucleolin/RNA interaction studies revealed a core binding sequence, corresponding to UAAAUC or AAAUCU. At the 3′-UTR, nucleolin assembles indirectly via protein/protein interaction, with the help of another 3′-UTR-binding protein, presumably annexin A2. The increased protein level of the ∼64-kDa nucleolin under hypoxia can be attributed to an autocatalytic cleavage of a high molecular weight nucleolin form, without alterations in nucleolin mRNA concentration. Thus, the alteration of translational efficiency by nucleolin, which occurs through a hypoxia inducible factor independent pathway, is an important step in C-P4H-α(I) regulation under hypoxia. Hypoxia is a pro-fibrotic stimulus, which is associated with enhanced collagen synthesis, as well as with augmented collagen prolyl 4-hydroxylase (C-P4H) activity. C-P4H activity is controlled mainly by regulated expression of the α C-P4H subunit. In this study we demonstrate that the increased synthesis of C-P4H-α(I) protein in human HT1080 fibroblasts under long term hypoxia (36 h, 1% oxygen) is controlled at the translational level. This is mediated by an interaction of RNA-binding protein nucleolin (∼64 kDa form) at the 5′- and 3′-untranslated regions (UTR) of the mRNA. The 5′/3′-UTR-dependent mechanism elevates the C-P4H-α(I) expression rate 2.3-fold, and participates in a 5.3-fold increased protein level under long term hypoxia. The interaction of nucleolin at the 5′-UTR occurs directly and depends on the existence of an AU-rich element. Statistical evaluation of the ∼64-kDa nucleolin/RNA interaction studies revealed a core binding sequence, corresponding to UAAAUC or AAAUCU. At the 3′-UTR, nucleolin assembles indirectly via protein/protein interaction, with the help of another 3′-UTR-binding protein, presumably annexin A2. The increased protein level of the ∼64-kDa nucleolin under hypoxia can be attributed to an autocatalytic cleavage of a high molecular weight nucleolin form, without alterations in nucleolin mRNA concentration. Thus, the alteration of translational efficiency by nucleolin, which occurs through a hypoxia inducible factor independent pathway, is an important step in C-P4H-α(I) regulation under hypoxia. Collagen prolyl 4-hydroxylase (C-P4H), 2The abbreviations used are: C-P4H, collagen prolyl 4-hydroxylase; RNP, ribonucleoproteins; HIF, hypoxia inducible factor; PH, prolyl hydroxylases; UTR, untranslated region; RT, reverse transcriptase; nt, nucleotide(s); hnRNP, heterogeneous nuclear ribonucleoprotein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.2The abbreviations used are: C-P4H, collagen prolyl 4-hydroxylase; RNP, ribonucleoproteins; HIF, hypoxia inducible factor; PH, prolyl hydroxylases; UTR, untranslated region; RT, reverse transcriptase; nt, nucleotide(s); hnRNP, heterogeneous nuclear ribonucleoprotein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. an α2/β2 tetramer, plays a central role in collagen synthesis. 4-Hydroxyproline residues are essential for the formation of triple-helical collagen molecules. The quantity and activity of C-P4H affects the composition of the extracellular matrix, because collagens constitute the major compound of extracellular matrix proteins. The collagen hydroxylation requires iron ions (Fe2+), 2-oxoglutarate, and oxygen (O2) (1Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar). Ascorbate is essential to maintain the iron ions in their biologically active Fe2+ form. Three different α-subunit containing C-P4Hs are known, resulting in the formation of three isoenzymes called C-P4H (I), (II), and (III) (2Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (321) Google Scholar). The β-subunit is identical to the enzyme and chaperone protein-disulfide isomerase (1Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar, 2Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (321) Google Scholar), and is required to keep the α-subunit in its soluble form (3Lumb R.A. Bulleid N.J. EMBO J. 2002; 21: 6763-6770Crossref PubMed Scopus (81) Google Scholar). The α-subunits contain the catalytical domains of the tetramer and are limiting in the formation of active P4Hs. Thus, P4H activity appears to be mainly regulated by the quantity of the α-subunit (4Kivirikko K.I. Helaakoski T. Tasanen K. Vuori K. Myllyla R. Parkkonen T. Pihlajaniemi T. Ann. N. Y. Acad. Sci. 1990; 580: 132-142Crossref PubMed Scopus (71) Google Scholar). The type (I) enzyme is the most abundant form of enzyme in most cells, except in chondrocytes and endothelial cells (5Annunen P. Autio-Harmainen H. Kivirikko K.I. J. Biol. Chem. 1998; 273: 5989-5992Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). However, enzymatic properties of types I–III isoenzymes are very similar (6Annunen P. Helaakoski T. Myllyharju J. Veijola J. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1997; 272: 17342-17348Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 7Kukkola L. Hieta R. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 47685-47693Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar).In addition to C-P4Hs, a second class of prolyl hydroxylases exist: the hypoxia inducible factor prolyl hydroxylases (HIF-PHs). They exclusively hydroxylate the transcription factor HIF (8Berra E. Ginouves A. Pouyssegur J. EMBO Rep. 2006; 7: 41-45Crossref PubMed Scopus (158) Google Scholar). Both families of PHs depend on oxygen, which is of tangible importance in various physiological (e.g. altitude) and pathophysiological (e.g. ischemia) settings. The mechanisms by which hypoxia induces gene transcription is well established (9Wenger R.H. Stiehl D.P. Camenisch G. Sci. STKE. 2005; 2005: re12PubMed Google Scholar). Hypoxia reduces activity of HIF-PHs that hydroxylate specific proline residues in the oxygen-dependent degradation domain of the HIF-1α subunit. As a consequence, HIF-1α accumulates and promotes hypoxic tolerance by activating gene transcription (10D'Angelo G. Duplan E. Boyer N. Vigne P. Frelin C. J. Biol. Chem. 2003; 278: 38183-38187Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The C-P4H-α(I) gene (P4HAI) is one of the genes, which is transcriptionally activated via HIF (11Takahashi Y. Takahashi S. Shiga Y. Yoshimi T. Miura T. J. Biol. Chem. 2000; 275: 14139-14146Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), but additionally, as postulated (12Fähling M. Perlewitz A. Doller A. Thiele B.J. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2004; 139: 119-126Crossref PubMed Scopus (35) Google Scholar) and as we investigated in detail in this work, it is also controlled posttranscriptionally. There seems to be a certain similarity to the collagens, which are also regulated at the posttranscriptional level (13Thiele B.J. Doller A. Kahne T. Pregla R. Hetzer R. Regitz-Zagrosek V. Circ. Res. 2004; 95: 1058-1066Crossref PubMed Scopus (60) Google Scholar). This coincidence may constitute a link between collagens and P4H as a basis of their co-regulation in collagen metabolism.Long term or chronic hypoxia is a strong stimulus for collagen synthesis resulting in organ fibrosis, in particular in heart and liver (14Falanga V. Martin T.A. Takagi H. Kirsner R.S. Helfman T. Pardes J. Ochoa M.S. J. Cell. Physiol. 1993; 157: 408-412Crossref PubMed Scopus (137) Google Scholar, 15Tajima R. Kawaguchi N. Horino Y. Takahashi Y. Toriyama K. Inou K. Torii S. Kitagawa Y. Biochim. Biophys. Acta. 2001; 1540: 179-187Crossref PubMed Scopus (13) Google Scholar). The accumulation of collagens in the extracellular matrix following hypoxia is mediated by transforming growth factor-β (16Falanga V. Zhou L. Yufit T. J. Cell. Physiol. 2002; 191: 42-50Crossref PubMed Scopus (140) Google Scholar, 17Papakonstantinou E. Aletras A.J. Roth M. Tamm M. Karakiulakis G. Cytokine. 2003; 24: 25-35Crossref PubMed Scopus (74) Google Scholar, 18Flanders K.C. Int. J. Exp. Pathol. 2004; 85: 47-64Crossref PubMed Scopus (513) Google Scholar), often associated with mRNA-specific posttranscriptional control (13Thiele B.J. Doller A. Kahne T. Pregla R. Hetzer R. Regitz-Zagrosek V. Circ. Res. 2004; 95: 1058-1066Crossref PubMed Scopus (60) Google Scholar). Post-transcriptional regulation, i.e. changes in mRNA stability or translational efficiency, is mainly attributed to the untranslated regions (UTRs) of mRNAs (19Dreyfuss G. Kim V.N. Kataoka N. Nat. Rev. Mol. Cell. Biol. 2002; 3: 195-205Crossref PubMed Scopus (1103) Google Scholar, 20Kuersten S. Goodwin E.B. Nat. Rev. Genet. 2003; 4: 626-637Crossref PubMed Scopus (423) Google Scholar, 21Keene J.D. Tenenbaum S.A. Mol. Cell. 2002; 9: 1161-1167Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar).The alteration of gene expression at the posttranscriptional level under stress conditions (22Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 2746-2753Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 23McGary E.C. Rondon I.J. Beckman B.S. J. Biol. Chem. 1997; 272: 8628-8634Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 24Spicher A. Guicherit O.M. Duret L. Aslanian A. Sanjines E.M. Denko N.C. Giaccia A.J. Blau H.M. Mol. Cell. Biol. 1998; 18: 7371-7382Crossref PubMed Scopus (56) Google Scholar), in particular by hypoxia, has been demonstrated for several genes including collagens (13Thiele B.J. Doller A. Kahne T. Pregla R. Hetzer R. Regitz-Zagrosek V. Circ. Res. 2004; 95: 1058-1066Crossref PubMed Scopus (60) Google Scholar), vascular endothelial growth factor (22Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 2746-2753Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 23McGary E.C. Rondon I.J. Beckman B.S. J. Biol. Chem. 1997; 272: 8628-8634Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 24Spicher A. Guicherit O.M. Duret L. Aslanian A. Sanjines E.M. Denko N.C. Giaccia A.J. Blau H.M. Mol. Cell. Biol. 1998; 18: 7371-7382Crossref PubMed Scopus (56) Google Scholar), erythropoietin (22Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 2746-2753Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 23McGary E.C. Rondon I.J. Beckman B.S. J. Biol. Chem. 1997; 272: 8628-8634Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 24Spicher A. Guicherit O.M. Duret L. Aslanian A. Sanjines E.M. Denko N.C. Giaccia A.J. Blau H.M. Mol. Cell. Biol. 1998; 18: 7371-7382Crossref PubMed Scopus (56) Google Scholar), and tyrosine hydroxylase (25Paulding W.R. Czyzyk-Krzeska M.F. J. Biol. Chem. 1999; 274: 2532-2538Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Recently it was shown that in response to hypoxia, more genes are regulated at the level of translation than by changes in transcription rates (26Koritzinsky M. Seigneuric R. Magagnin M.G. van den B.T. Lambin P. Wouters B.G. Radiother. Oncol. 2005; 76: 177-186Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Recent studies revealed that C-P4H-α(I) mRNA may belong to a subclass of transcripts that are characterized by an increased translational efficiency under hypoxia (11Takahashi Y. Takahashi S. Shiga Y. Yoshimi T. Miura T. J. Biol. Chem. 2000; 275: 14139-14146Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 12Fähling M. Perlewitz A. Doller A. Thiele B.J. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2004; 139: 119-126Crossref PubMed Scopus (35) Google Scholar). The aim of this study was to analyze the molecular mechanism in the regulation of C-P4H-α(I) expression under hypoxia. In particular, we addressed the role of the 5′- and 3′-UTRs of C-P4H-α(I) mRNA in this process.EXPERIMENTAL PROCEDURESCell Culture and RNA/Protein IsolationHuman fibrosarcoma HT1080 (ATCC, passages 16–21) cells were maintained in Dulbecco's modified Eagle's medium (high glucose; PAA Laboratories GmbH), supplemented with 10% heat-inactivated fetal calf serum, 50 units/ml penicillin, 50 μg/ml streptomycin, 15 mm Hepes, and 2 mmol/liter glutamine, at 37 °C, 5% CO2. Before use in experiments, cells were maintained in a medium containing 0.4% fetal calf serum for at least 24 h. Measurements started with the application of fresh medium containing 0.4% fetal calf serum. For hypoxic conditions the cells were incubated in a hypoxic chamber (JOUAN IG750). Oxygen content was reduced to 1% by gas exchange with 95% nitrogen, 5% CO2. Control cells were incubated under atmospheric oxygen conditions (21% O2, 5% CO2, 37 °C). To inhibit translation, cells were incubated in the presence of cycloheximide (20 μg/ml). For RNA and protein isolation, cells were washed with ice-cold phosphate-buffered saline. RNA was prepared using RNA-Bee (Biozol Diagnostica Vertrieb GmbH) according to the manufacturer's protocol. Protein extracts (10,000 × g supernatants, S10) were isolated using lysis buffer (10 mm Tris, pH 7.5, 140 mm NaCl, 1 mm EDTA, 25% glycerol, 0.1% SDS, 0.5% Nonidet P-40, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1× Complete protease inhibitor mixture; Roche Diagnostics).mRNA QuantificationRT-PCR—mRNA levels quantified by RT-PCR were normalized to relative β-actin levels. Primers were designed to bridge at least one intron. PCR conditions were used as follows: 3 min at 95 °C, cycles were 30 s at 95 °C, 30 s of annealing, 30 s at 72 °C, final elongation was for 2 min at 72 °C; 2.5 mm MgCl2. The primers were as follows: C-P4H-α(I), forward, 5′-CCACAGCAGAGGAATTACAG, reverse, 5′-ACACTAGCTCCAACTTCAGG; β-actin, forward, 5′-TGAAGTGGTACGTGGACATC, reverse, 5′-GTCATAGTCCGCCTAGAAGC; nucleolin, forward, 5′-AGACAGAAGCTGATGCAGAG, reverse, 5′-TGTTGCACTGTAGGAGAGGT.Northern Blotting—Isolated RNA was separated by electrophoresis on 1% agarose gels containing formaldehyde. The RNA was capillary transferred to positively charged nylon membranes (Roche Diagnostics), visualized after ethidium bromide staining to document the relative level of 18 S and 28 S rRNA, and hybridized to digoxigenin-labeled partial C-P4H-α(I) antisense transcripts (1,600 nt, representative for the coding region). The detection was performed using the digoxigenin RNA Labeling Kit (Roche Diagnostics) according to the manufacturer's protocol. mRNA levels were normalized to 18 S/28 S rRNA.Estimation of mRNA StabilitymRNA stability assays were performed as described in Ref. 27Fähling M. Mrowka R. Steege A. Martinka P. Persson P.B. Thiele B.J. J. Biol. Chem. 2006; 281: 9279-9286Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar.Differential CentrifugationFor investigation of mRNA and protein localization, cells were incubated in the presence of cycloheximide (20 μg/ml) for 10 min. Cells were washed in ice-cold phosphate-buffered saline supplemented with cycloheximide (20 μg/ml), harvested, and cell extracts were prepared using lysis buffer 2 (20 mm Tris, pH 7.5, 150 mm KCl, 25 mm MgCl2, 0.25% Nonidet P-40, 200 units/ml RNase OUT (Invitrogen), 20 μg/ml cycloheximide, 1× Complete protease inhibitor mixture (Roche Diagnostics)). After a 10-min incubation on ice cells were centrifuged at 1,000 × g at 4 °C for 10 min. Supernatants were subjected to ultracentrifugation at 100,000 × g at 4 °C, for 2 h. Sediments represent a translationally active fraction. Supernatants represent a polysome-free fraction. After differential centrifugation, RNA was extracted from sediments and supernatants using RNA-Bee and analyzed by Northern blotting. For determination of protein localization, sediments were resolved in equal amounts of lysis buffer 2, and analyzed by Western blotting.Western BlottingProtein extracts (30 μg/sample) were separated by SDS-PAGE. After electrophoresis, proteins were transferred to Hybond™-P membranes (Amersham Biosciences) using a Bio-Rad Mini Trans-Blot transfer cell. The membranes were blocked for 1 h with 5% Blot-Quick Blocker (Chemicon). Following the blocking step, the membranes were incubated in 1% blocking solution containing a primary antibody (anti-P4H-α antibody, Acris Antibodies GmbH; anti-nucleolin antibody, Santa Cruz Biotechnology Inc.; anti-annexin A2 antibody, Acris Antibodies GmbH) at room temperature for 1.5 h or overnight at 4 °C. The membranes were washed three times with Tris-buffered saline with Tween 20 and incubated with a secondary antibody (anti-mouse, Promega) for 1 h. After additional washing steps bands were detected using the ChemiGlow™-West Detection Kit (Alpha Innotech Corporation). Membranes were stripped for 5 min with distilled water, 5–15 min in 0.2 m NaOH, and 5 min in distilled water and reprobed with anti-α-actin (Chemicon) or anti-glyceraldehyde-3-phosphate dehydrogenase (Acris Antibodies GmbH) antibodies to detect relative β-actin and glyceraldehyde-3-phosphate dehydrogenase levels as loading control.Molecular Cloning and in Vitro TranscriptionPartial C-P4H-α(I) sequences (GenBank™ gi:63252885), representing the C-P4H-α(I) 5′-UTR (133 nt) and 3′-UTR (999 nt) were amplified by PCR, cloned, and transformed using the TOPO®II TA Cloning® Kit (Invitrogen). Positive clones were confirmed by sequencing. For RNA/protein interaction studies, sense transcripts, representing the 5′- or 3′-UTR of C-P4H-α(I) mRNA were prepared as described above and transcribed using the T7-polymerase. In vitro transcripts were purified by BD Chroma Spin™-100 (DEPC) columns (Clontech).UV Cross-linkingIn vitro transcripts representing the 5′- or 3′-UTRs of C-P4H-α(I) mRNA were radioactively labeled using [α-32P]uridine-, [α-32P]cytosine-, [α-32P]adenine-, or [α-32P]guanosine 5′-triphosphate (800 Ci/mmol, MP Biomedicals Germany GmbH).UV Cross-linking Experiments1–2 ng representing 100,000 cpm of [α-32P]UTP-labeled in vitro transcripts were incubated with 35 μg of cytosolic protein extract for 30 min at room temperature in 10 mm Hepes, pH 7.2, 3 mm MgCl2, 5% glycerol, 1 mm dithiothreitol, 150 mm KCl, and 2 units/μl RNase OUT (Invitrogen) in the presence of rabbit rRNA (0.5 μg/μl). Then the samples were exposed to UV light (255 nm, 1.6 joule, UV Stratalinker) on ice, treated with RNase A (30 μg/ml final concentration) and RNase T1 (750 units/ml final concentration) for 15 min at 37 °C and subjected to 12% SDS-PAGE and autoradiography. For competition assays, a 50-fold excess of unlabeled in vitro transcripts was added. For cis-element analyses by separate labeling of the four possible nucleotides the radioactive activity of all nucleotides was adjusted to comparable levels before in vitro transcription. Equal RNA concentrations (2 ng) of the resulting in vitro transcripts were used for the UV cross-linking assay.Mapping the Nucleolin Binding Motif in C-P4H-α(I) 5′-UTR using a Mathematical ApproachRelative amounts, r[A,C,G,U], of UV cross-linking signals, corresponding to ∼64-kDa nucleolin, were scanned and statistically evaluated. The signals result from the label transfer of separate radioactively labeled [α-32P]uridine-, [α-32P]cytosine-, [α-32P]adenine-, or [α-32P]guanosine 5′-triphosphate in vitro transcripts to nucleolin. The signal intensity depend on the qualitative and quantitative composition of each nucleotide in the RNA/protein interaction site. To map the protein-related intensity pattern of the relative amount, r[A,C,G,U], of the cross-linking experiments to a sequence motif in the 5′-UTR of the P4H-α(I) mRNA the following algorithm was applied. A sliding window of 6 nt was shifted over the 5′-UTR. For each position, p, the relative theoretical amount a[A,C,G,U],p of each nucleotide in the window was determined.Our mapping score is the inverse of an error function between the theoretical and the measured value within a sliding window. The error function calculates the sum of the squared differences of the theoretical and measured nucleotide fraction. At each position p the mapping score was determined according the following equation.ms(p)=1(rA-aA,p)2+(rC-aC,p)2+(rG-aG,p)2+(rU-aU,p)2 A high value of ms(p) corresponds to a high probability of the motif matching the quantified radioactively labeled pattern. Possible effects of the neighborhood are considered by the experimental design, i.e. in vitro transcripts were similar in size and sequence. Hence, the influence of neighboring sequences as well as secondary structure are considered in the assay.Affinity ChromatographyFor the isolation of mRNA-binding proteins, in vitro transcripts representing the 5′- or 3′-UTR of C-P4H-α(I) mRNA were generated in the presence of biotinylated CTP (Invitrogen). Cytosolic extracts (5 mg protein) were incubated with 1 μg in vitro labeled transcript for 30 min at room temperature. RNP (ribonucleoprotein) complexes were isolated using 200 μl of streptavidin-agarose/sample (Sigma). Samples without the addition of biotinylated transcripts served as negative control. The agarose beads were centrifuged for 15 s at 5,000 × g and washed six times (20 mm Tris, pH 7.4, 150 mm KCl, 3 mm MgCl2, 0.5 mm dithiothreitol). The last two washing supernatants were used as control. The RNP complexes were eluted using high salt buffer (20 mm Tris, pH 7.4, 2 m KCl, 3 mm MgCl2, 0.5 mm dithiothreitol). Proteins were precipitated, solved, and subjected to SDS-PAGE. After Coomassie staining protein signals representing specific RNA-binding factors were excised. Tryptic digestion of proteins was carried out using ZipPlates (Millipore) without reduction or alkylation. Tryptic fragments were analyzed by Reflex IV MALDI-TOF mass spectrometer (Bruker-Daltonics). Mass spectra were analyzed using Mascot software 2.0 with automatic searches in NCBI nonredundant databases. Search parameters allowed for one miscleavage and oxidation of methionine. Criteria for positive identification of proteins with MS were set according to the scoring algorithm delineated in Mascot (28Pappin D.J. Hojrup P. Bleasby A.J. Curr. Biol. 1993; 3: 327-332Abstract Full Text PDF PubMed Scopus (1416) Google Scholar).Reporter Gene ConstructsFor reporter gene assays the Luciferase vector pGL3-promotor (Promega, constitutive SV40 promoter) was modified. The vector-specific 5′- and 3′-UTRs of luciferase mRNA were replaced by the human C-P4H-α(I) UTRs. The UTRs were amplified by PCR and restriction sites were added by primer extension. The 5′-UTR of P4H-α(I) mRNA was cloned using the pGL3p vector-specific HindIII and NcoI restriction sites and the 3′-UTR (including the poly-A signal) using the XbaI and BamHI restriction sites. Artificial 3′-UTR parts represent the first 500 nt (3′A) and the terminal 522 nt (3′B) of the 3′-UTR and were designed to overlap by 20 nt. For the 5′-UTR mutations a partial 5′ sequence was amplified and the mutated element was added by primer extension. The quality of processed vectors was confirmed by sequencing. The resulting vector constructs expressed a constitutively transcribed luciferase transcript with or without the specific C-P4H-α(I) UTRs.Reporter Gene AssaysHT1080 cells were cultured in 96-well plates (μClear Platte 96K, Greiner BIO-ONE GmbH) and co-transfected with the firefly luciferase pGL3-promotor vector (Promega), as well as its transformed variants, and the Renilla luciferase phRL-TK vector (ratio 1:3) using the FuGENE 6 Transfection Reagent (Roche Diagnostics Corp.) according to the manufacturer's protocol. After 6 h, the transfection medium was removed, and measurements were started after adding fresh medium. The luciferase activity was detected using a luminometer (Labsystems Luminoscan RS) programmed with individual software (Luminoscan RII, Ralf Mrowka). The transfection with the Renilla luciferase served as a control.Statistical AnalysisAutoradiographic signals were scanned and quantified using the Scion Image software (Scion Corp.). Results appear as means, and error bars represent the standard deviation (S.D.). Data were analyzed using the Student's t test, and the null hypothesis was rejected at the 0.05 level.RESULTSPost-transcriptional Regulation of C-P4H-α(I) Expression under Long Term Hypoxia—Cell culture experiments, using human fibrosarcoma HT1080 cells, clearly demonstrate that C-P4H-α(I) is induced at the mRNA and protein levels under hypoxia (1% oxygen) (Fig. 1). Interestingly, during the late phase of a time scale up to 36 h C-P4H-α(I) protein increases independently of the mRNA concentration, suggesting a posttranscriptional component in the mechanism of expression control. We observed ∼2-fold elevated mRNA and protein levels after 10 h hypoxia, compared with control. Whereas the mRNA concentration remained relatively constant also under long term hypoxic conditions (up to 36 h) and even dropped slightly, the protein level increased continuously. Under long term conditions C-P4H-α(I) protein levels increased nearly 6-fold, compared with a less than 3-fold increase at the mRNA level. The elevated mRNA level can be attributed to the transcriptional action of HIF (11Takahashi Y. Takahashi S. Shiga Y. Yoshimi T. Miura T. J. Biol. Chem. 2000; 275: 14139-14146Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Consistently, we did not observe significant alterations of the C-P4H-α(I) mRNA stability (Fig. 2, A and B). The significantly stronger increase seen at the protein level may have two reasons: either protein degradation was inhibited or translational efficiency was increased. Inhibition of translation by cycloheximide prevented the increase of the protein level by hypoxia, indicating that the elevated protein concentration was due to newly synthesized protein (Fig. 2, C and D). Furthermore, the C-P4H-α(I) protein level dropped to about one-third by cycloheximide under both, hypoxic and atmospheric conditions. This suggests that the rate of protein degradation was not affected by hypoxia.FIGURE 2C-P4H-α(I) mRNA and protein stability under hypoxia. HT1080 cells were incubated under control or hypoxic conditions for 24 h. A, cytosolic extracts were isolated as described in Ref. 27Fähling M. Mrowka R. Steege A. Martinka P. Persson P.B. Thiele B.J. J. Biol. Chem. 2006; 281: 9279-9286Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar and incubated at room temperature up to 4 h. mRNA levels were estimated by RT-PCR. The increased C-P4H-α(I) mRNA level under hypoxia was adapted to the 0 h control level by reduction of PCR cycles. The time-dependent mRNA decay is indicated by the decreased mRNA level. A pool of six independent samples is shown. β-Actin mRNA levels (half-life time about 4 h) are shown as a comparison and were not changed significantly. B, graphic display of mRNA decay of C-P4H-α(I) mRNA under control conditions (half-life time 1.82 h ± 0.41 S.D.), compared with hypoxia (half-life time 2.1 h ± 0.28 S.D.) (n = 6). There is no significant alteration in the C-P4H-α(I) mRNA stability under hypoxia. C and D, cycloheximide (20 μg/ml) was used to inhibit the translation of proteins. C-P4H-α(I), β-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels were visualized by the Western blotting technique. A representative set of data are shown and was statistically evaluated (n = 6). 24-h cycloheximide treatment reduced the C-P4H-α(I) level to one-third, which is similar under both, control and hypoxic conditions.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether C-P4H-α(I) is regulated at the posttranscriptional level, we generated reporter gene constructs. For this purpose 5′- and 3′-UTRs of luciferase mRNA were replaced by specific 5′- and/or 3′-UTRs of C-P4H-α(I) mRNA (for a schematic illustration see Fig. 3A), and reporter gene assays were performed after transient transfection. The transcription rate of the reporter gene was controlled by a constitutive SV40 promoter. Thus, differences in luciferase activity depended solely on the regulatory capacity mediated by the C-P4H-α(I) UTRs. Long term hypoxia (36 h) did not influence the luciferase expression/activity resulting from the original reporter gene. The replacement of the original 5′-UTR by the C-P4H-α(I) 5′-UTR also showed no significant changes, whereas the replacement of the 3′-UTR led to a 1.3-fold increased luciferase activity. Interestingly, the combination of C-P4H-α(I) 5′- and 3′-UTRs potentiated the effect: expression reached a 2.2-fold level versus control (Fig. 3B), which correlated well with the discrepancy between mRNA and the protein level under long term hypoxia. Dividing the 3′-UTR into two parts (termed 3′A and 3′B) did not result in a comparable activation (Fig. 3C). Neither the individual 5′∼500 nt, nor the terminal 3′∼500 nt of the 3′-UTR reached the full activity of the complete 3′-UTR. Obviously, 3′-UTR parts did not represent the properties of the complete 3′-UTR. This is in contrast to findings after Fe2+ diminishment, where the regula" @default.
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