Mast cells are derived from multipotential hematopoietic stem cells, circulate in blood as immature cells, and undergo the final stages of their differentiation and maturation after their agranular progenitors become lodged in tissues(1, 2, 3, 4, 5) . As assessed by their expression of three different classes of granule proteases, at least four phenotypically different types of mast cells are present in the tissues of the BALB/c mouse. Mast cells express at least four chymotrypsin-like neutral proteases (designated chymases) whose genes reside at a complex on chromosome 14(6) , along with the genes that encode cathepsin G and granzymes B, C, E, and F(7, 8, 9) . Mast cells in the intestinal mucosa preferentially express mouse mast cell protease (mMCP) ()1 and mMCP-2(5, 10, 11, 12, 13, 14) , the mast cells in the peritoneal cavity and skin preferentially express mMCP-4 and mMCP-5(15, 16, 17) , and those in the spleen express every known granule chymase(5) .

Based on in vitro studies, it appears that much of the granule protease pleiotropism of the mast cell is cytokine regulated. The immature mast cells (mBMMC) generated by culturing BALB/c mouse bone marrow cells in interleukin (IL) 3-enriched medium(18, 19, 20) contain high steady-state levels of the transcripts that encode mMCP-5 (17) but not mMCP-1(13) , mMCP-2(12) , or mMCP-4(16) . However, these mBMMC will also express high steady-state levels of the mMCP-1 and mMCP-2 transcripts when cultured in the presence of IL-9 (21) or IL-10 (14, 22) , or high steady-state levels of the mMCP-4 transcript when cultured in the presence of c-kit ligand(23) . Furthermore, the steady-state levels of the mMCP-1 and mMCP-2 transcripts in BALB/c mBMMC can be reversibly altered by adding or withdrawing IL-10 from the culture medium(14, 22) . Half-maximal levels of both chymase transcripts are achieved after the cells are cultured with IL-10 for only 24 h, and plateau levels of the two chymase transcripts are reduced to half-maximum within 24 h of IL-10 withdrawal. The rate by which the steady-state levels of the two protease transcripts in these nontransformed mast cells can be increased or decreased raised the possibility that post-transcriptional control mechanisms might contribute to mast cell protease heterogeneity.

We now demonstrate that the IL-10-regulated expression of the chymase mMCP-2 in BALB/c mBMMC occurs primarily by a post-transcriptional mechanism. This discovery has broad implications for understanding how the expression of the chromosome 14 superfamily of serine proteases is regulated in mast cells and other hematopoietic cells during inflammation.

EXPERIMENTAL PROCEDURES

Cell Culture

RNA Blot and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analyses

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RT-PCRs were performed with total or cytoplasmic RNA isolated from 3-6-week-old BALB/c mBMMC. Samples were incubated with RNase-free DNase I for 10 min at room temperature to remove residual genomic DNA. Using the cDNA cycle kit from Invitrogen (San Diego, CA), 2 µg of RNA and 0.2 µg of oligo(dT) primer (18-mer) were placed in a mirocentrifuge tube and incubated at 65 °C for 10 min to disrupt the secondary structure of the transcript. Each RNA sample was reverse-transcribed at 42 °C for 1 h in 20 µl of a solution containing 1 reverse transcription buffer, 10 units of RNase inhibitor, 20 mM deoxynucleotide triphosphates, 20 mM sodium pyrophosphate, and 5 units of avian myeloblastosis virus reverse transcriptase. The reaction mixture was extracted with 20 µl of phenol-chloroform, precipitated with 2 volumes of ethanol, and resuspended in 20 µl of distilled water. A 2-µl sample of the resulting cDNA preparation was mixed with 48 µl of 1 PCR buffer containing 2 mM MgCl, 2.5 units of AmpliTaq polymerase (Perkin-Elmer), 0.2 µg of a primer corresponding to a 5` region in exon 1 (5`-ACTGGCAAAATGCAGGCC-3`) of the mMCP-2 transcript, and 0.2 µg of a primer corresponding to a 3` region in exon 5 (5`-CATCATCACAGACATGTG-3`) of the mMCP-2 transcript. Thirty to 35 cycles of PCR were performed. Each cycle consisted of a 1-min denaturation step at 94 °C, a 2-min annealing step at 55 °C, and a 3-min extension step at 72 °C. To verify the authenticity of their nucleotide sequences, the RT-PCR products were purified by electrophoresis on a 1% agarose gel. The appropriate bands were excised, purified with Geneclean(TM) (BIO 101, Vista, CA), and defined by Taq cycle-sequencing technology at the Biocore Facility of the Dana Farber Cancer Institute.

Nuclear Run-on Assays

mBMMC (1-5 10) maintained in medium containing or lacking IL-10 were washed with cold phosphate-buffered saline and disrupted by two treatments with 5 ml of ice-cold lysis buffer (10 mM NaCl, 3 mM MgCl, 0.5% Nonidet P-40, and 10 mM Tris-HCl, pH 7.4). After centrifugation at 800 g for 5 min at 4 °C, the nuclei-enriched pellets were resuspended in 200 µl of storage buffer (40% glycerol, 5 mM MgCl, 0.1 mM EDTA, and 50 mM Tris-HCl, pH 7.5). Each nuclear run-on reaction was initiated in a 400-µl reaction buffer containing 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 1 mM dithiothreitol, 10 units of RNase inhibitor, 100 µCi of [P]UTP, 20% glycerol, 5 mM MgCl, 150 mM KCl, and 30 mM Tris-HCl, pH 8.0. The reaction mixture was incubated at 30 °C for 30 min and then for 5 min at room temperature with 25 µg/ml DNase I (RNase-free). Tri Reagent (750 µl) and CHCl (200 µl) were added, and the radiolabeled nascent nuclear RNAs in the aqueous phase were precipitated with isopropanol, resuspended in 100 µl of buffer (10 mM Tes, 10 mM EDTA, and 0.2% SDS), purified with a gel filtration spin column (Clontech, Palo Alto, CA) and incubated with one of the DNA slot blots. Each hybridization reaction was carried out at 65 °C for 36-48 h in a small vial containing 1-2 ml of hybridization buffer (10 mM Tes, 10 mM EDTA, 0.2% SDS, 0.6 NaCl, 1 Denhardt's solution, and 300 µg/ml salmon sperm DNA) and 1 to 5 10 cpm of radiolabeled nuclear RNA. After hybridization, the blot was treated with 10 µg/ml RNase A for 20 min at 37 °C, washed three times (20 min each) with 2 SSC at 65 °C, and exposed to x-ray film for 1-3 days.

Plasmids containing either a full-length -actin, mMCP-2, or mMCP-4 cDNA were also digested with a restriction enzyme, and the digests (10 µg of DNA/lane) were electrophoresed in a 1.2% agarose gel. The separated DNA was transferred to a nylon membrane, and the resulting blot was evaluated for hybridization to the appropriately sized insert with a replicate sample of radiolabeled nuclear RNA.

Pulse-Chase Experiments

Effects of Actinomycin D and Cycloheximide on the Steady-state Levels of the mMCP-2 Transcript

To determine if an inhibitor of protein synthesis can block the IL-10-induced expression of the mMCP-2 transcript, mBMMC (10) were washed and resuspended in 50% WEHI-3 cell-conditioned medium containing IL-10 and varying doses (0, 1, 5, and 20 µg/ml) of cycloheximide (Sigma). After 24 h of culture, total RNA was isolated from the four populations of mBMMC, and the amount of mMCP-2 mRNA in each was quantitated by blot analysis as described above. To determine if cycloheximide can influence the steady-state level of the established mMCP-2 transcript, 10 mBMMC were pretreated with IL-10, washed in enriched medium, and resuspended in 50 ml of 50% WEHI-3 cell-conditioned medium containing 1 µg/ml cycloheximide with or without IL-10. At various time points, total RNA was isolated and the steady-state levels of the mMCP-2 transcript in the resulting 10 samples were quantitated by blot analysis.

RESULTS

Transcription of Chymase Genes

Figure 1: Transcription of chymase genes. A, for analysis of the steady-state levels of chymase transcripts, a blot containing total RNA from BALB/c mBMMC cultured in the absence(-) or presence (+) of IL-10 for 36 h was analyzed sequentially with mMCP-1, mMCP-2, mMCP-4, mMCP-5, and -actin gene-specific probes. B, for nuclear run-on analysis, BALB/c mBMMC were cultured for 24 h in the absence(-) or presence (+) of IL-10. Isolated nuclear run-on derived transcripts were incubated with a membrane containing plasmid DNA alone (pBS) or plasmid DNA carrying a gene-specific probe for mMC-CPA, type IX collagen (ColIX), mMCP-1, mMCP-2, mMCP-4, mMCP-5, and -actin. pBS and type IX collagen plasmids were used as negative controls; -actin and mMC-CPA plasmids were used as positive controls. Similar findings were obtained in four other experiments.

RT-PCR and Nucleotide Sequence Analysis of the mMCP-2 Transcript

Figure 2: RT-PCR analysis of mMCP-2 transcripts. Samples of total RNA from BALB/c mBMMC cultured in the presence (lane 1) or absence (lane 2) of IL-10 were subjected to RT-PCR (30 cycles) with a set of primers specific for mMCP-2. The resulting products were then assessed for their relative size by agarose gel electrophoresis. The arrow on the left indicates the 910-bp product expected if the mature mMCP-2 transcript is present in a RNA sample. Size markers are indicated in lane 3 and on the right. Similar findings were obtained in four other experiments with other sets of mMCP-2-specific primers.

Determination of the Half-life of the mMCP-2 Transcript by Pulse-Chase Analysis

Figure 3: Stability of the mMCP-2, mMCP-5, and -actin transcripts. mBMMC were radiolabeled with [H]uridine in the presence of IL-10 and then were washed and cultured in radioactive-free medium with () or without () IL-10. The amounts of radiolabeled mMCP-2, mMCP-5, and -actin transcripts in each culture were determined at the indicated time points in the chase. Results are expressed as a percent of the starting level of the transcript. In these experiments, the specific binding of radiolabeled transcripts to the mMCP-2 probe was 17-81-fold higher than the background binding of radiolabeled transcripts to pBS.

Effect of Actinomycin D and Cycloheximide on the Steady-state Levels of the mMCP-2 Transcript

Figure 4: Effect of actinomycin D on the steady-state levels of the mMCP-2 transcript. BALB/c mBMMC, cultured in the presence of IL-10 to induce high steady-state levels of the mMCP-2 transcript, were washed and cultured in the presence of actinomycin D (Act D) with (+) or without(-) IL-10. At the indicated time points, RNA was isolated and analyzed for the presence of the mMCP-2 transcript (top panel). The change in the level of the mMCP-2 transcript is shown in the bottom panel as a percent of the starting level of the transcript. The RNA gel used in the depicted experiment was also stained with ethidium bromide (middle panel) to demonstrate that comparable amounts of 18 S ribosomal RNA were loaded into the lanes. Similar findings were obtained in two other experiments.

To determine whether or not the IL-10-regulated expression of the mMCP-2 transcript in BALB/c mBMMC requires de novo protein synthesis, cells were treated with IL-10 for 24 h in the absence or presence of 1 to 20 µg/ml cycloheximide. The IL-10-induced expression of the mMCP-2 transcript was effectively blocked by concomitant exposure of the cells to 1 µg/ml cycloheximide (Fig. 5A). In a control experiment, when mBMMC were cultured in WEHI-3 cell-conditioned medium containing IL-10 and 1 µg/ml cycloheximide for 24 h, washed, and then cultured in the presence of IL-10, but in the absence of cycloheximide for 48 h, the cells regained their ability to express high steady-state levels of the mMCP-2 transcript (data not shown). Thus, the cycloheximide effect on the steady-state level of the mMCP-2 transcript in mBMMC is reversible.

Figure 5: Effect of cycloheximide on the steady-state levels of mMCP-2 mRNA. A, BALB/c mBMMC were cultured for 24 h in medium containing IL-10 without (lane 1) or with 1 (lane 2), 5 (lane 3), or 20 (lane 4) µg/ml cycloheximide (CHX). An RNA blot prepared from these cells was analyzed for the presence of mMCP-2 mRNA (top panel) and 18 S ribosomal RNA (bottom panel). Similar findings were obtained in a preliminary experiment with 20 µg/ml cycloheximide. B, BALB/c mBMMC were cultured in medium containing IL-10 for 48 h and then were resuspended in medium containing both IL-10 and cycloheximide (lanes 4, 6, 8, and 10) or just cycloheximide (lanes 3, 5, 7, and 9). RNA was isolated from the various populations of mBMMC immediately (lanes 3 and 4), or at 8 h (lanes 5 and 6), 16 h (lanes 7 and 8), and 32 h (lanes 9 and 10) after the initiation of the cycloheximide treatment. For controls, replicate mBMMC were cultured in medium containing IL-10 but not cycloheximide (lane 1) or in medium containing neither IL-10 nor cycloheximide (lane 2). A blot was prepared and analyzed for the presence of the mMCP-2 transcript (top panel) and 18 S ribosomal RNA (middle panel). Results are expressed in the bottom panel as a percent of the starting level of the transcript. Similar findings were obtained in another experiment.

To determine if the half-life of the mMCP-2 transcript in IL-3-developed mBMMC depends on the synthesis of protein, mBMMC were exposed to IL-10 for 48 h to induce high steady-state levels of the mMCP-2 transcript. The cells were washed and then cultured for up to 32 h in WEHI-3 cell-conditioned medium containing 1 µg/ml cycloheximide with or without IL-10. The steady-state levels of the mMCP-2 transcript decreased in the two populations of mBMMC at comparable rates (Fig. 5B).

DISCUSSION

Mast cell heterogeneity is now well established in the mouse, rat, and human, and the chymase superfamily of granule proteases has served as the principal marker for identifying tissue-specific mast cell phenotypes in these three species. Because the individual members of the chymase superfamily are differentially regulated in cultured mouse mast cells by cytokines, we examined how the expression of the chymase mMCP-2 is controlled in BALB/c mBMMC. We now report that the expression of mMCP-2 in these mast cells is regulated primarily by a post-transcriptional mechanism.

Based on the amino acids in their putative substrate binding pockets (17, 34) , it is likely that each mast cell chymase whose gene resides at the chromosome 14 complex (6) has a preferred substrate in vivo. In terms of their deduced amino acid sequences and the nucleotide sequences of their genes and transcripts, mMCP-1, mMCP-2, and mMCP-4 are more similar to one another than to mMCP-5. BALB/c mBMMC developed with recombinant IL-3- or IL-3-enriched conditioned medium contain high steady-state levels of the mMCP-5 transcript, but the levels of the transcripts that encode the other members of the chymase superfamily are all below detection by RNA blot analysis (Fig. 1A and Refs. 12, 13, 16, and 17). Nonetheless, we now show by nuclear run-on analysis with gene-specific probes that all chymase genes are transcribed in these BALB/c mBMMC (Fig. 1B). Although it is possible that differential rates of transcription contribute somewhat to the overall steady-state levels of cytokine-inducible chymase transcripts, the nuclear run-on data indicate that the levels of these transcripts are controlled primarily by a post-transcriptional mechanism. When mBMMC cultured in the absence of IL-10 were compared to mBMMC cultured in the presence of IL-10, there was not much difference in the rates of transcription of the mMCP-1 and mMCP-2 genes despite the fact that there was a vast difference in the steady-state levels of these two chymase transcripts. Furthermore, even though the mMCP-4 gene is transcribed at a high rate with or without IL-10-stimulation, no mMCP-4 transcript could be detected in these cells by RNA blot analysis. RT-PCR analysis (Fig. 2) confirmed that the transcript that hybridizes to the mMCP-2 cDNA in the nuclear run-on analysis is mMCP-2. Moreover, nucleotide sequence analysis of the RT-PCR product revealed that the mMCP-2 transcript is properly processed in mBMMC whether these cells are cultured in the absence or presence of IL-10, a cytokine that induces high steady-state levels of mMCP-2 mRNA in these mast cells.

Because the levels of the mMCP-1, mMCP-2, or mMCP-4 transcripts in IL-3-developed mBMMC are below detection by blot analysis (Fig. 1A), the post-transcriptional control of any one of these mMCP transcripts can be studied only after its expression has been induced by cytokine treatment of the cells. We previously reported (14) that if IL-10-stimulated mBMMC are subsequently cultured in the absence of IL-10, there is a slow, but steady, decrease in the steady-state level of the mMCP-2 transcript. Pulse-chase experiments revealed that the mMCP-2 transcript has an 4-fold longer half-life in IL-10-treated mBMMC that continue to be cultured in the IL-10-enriched medium than in replicate cells that have been transferred into IL-10-free medium (Fig. 3). IL-10 did not influence the half-life of the mMCP-5 or -actin transcripts in these cells. However, the acquisition of high steady-state levels of the mMCP-2 transcript in IL-10-treated cells was fully blocked by cycloheximide (Fig. 5A), indicating that the inductive phase of the regulation depends on the synthesis of new protein. Inasmuch as the mMCP-2 gene is transcribed in mBMMC that have been cultured in the absence of IL-10 (Fig. 1B and Fig. 2), IL-10 must induce synthesis of a trans-acting protein that directly or indirectly stabilizes the mature mMCP-2 transcript or the processing of its precursor. If the mMCP-2 transcript is already expressed in abundance in mBMMC, the half-life of the transcript decreases from a rate of 34 h in nondrug-treated cells (Fig. 3) to 13-15 h in cells treated with actinomycin D (Fig. 4) or cycloheximide (Fig. 5B). These data support the conclusion that IL-10 induces expression of a trans-acting factor that stabilizes the mMCP-2 transcript.

The fact that the half-life of the mMCP-2 transcript in cells exposed to IL-10 and actinomycin D (Fig. 4) or cycloheximide (Fig. 5B) does not decrease to 8 h could be a consequence of a secondary drug effect on the catabolism machinery itself. Alternatively, both drugs could effect the movement and processing of newly synthesized mMCP-2 pre-mRNA, as has been found with the IL-2 precursor transcript in cycloheximide-treated tonsil cells(35) . If IL-10 induces the expression of a protein that stabilizes the mMCP-2 transcript, then the rate of turnover of the mMCP-2 transcript in the pulse-chase assay (Fig. 3) depends, in part, on the rate of turnover of the stabilizing protein. Because time would be required to deplete the intracellular level of the stabilizing protein following the removal of IL-10 from the culture medium, the 4-fold difference in the turnover of the mMCP-2 transcript in the two populations of mBMMC is a minimum estimate. It, therefore, is likely that the rate of decay of the mMCP-2 transcript is extremely rapid in IL-3-developed mBMMC that have never been stimulated with IL-10.

The half-life of a transcript, which can differ widely in cells, depends on the presence or absence of certain structural features in the transcript and trans-acting factors in the cell(36) . Some differentiating cells respond to developmental signals by changing the stability of their transcripts. For example, during erythrocyte development, the accumulation of globin is attributed mainly to the preferential stabilization of globin mRNA(37) . When progenitor cells differentiate into mature B lymphocytes, high levels of immunoglobulin are produced due to increased stabilization of the immunoglobulin transcript(38) . mBMMC are nontransformed mast cell-committed progenitors. The post-transcriptional regulatory pathway for protease expression in these cells may reflect their granule immaturity.

It is known that cis-acting elements in the 3`-UTRs of numerous transcripts regulate their half-life in cells. The most thoroughly studied RNA motif that controls the steady-state levels of mRNA in cells is the adenylate- and uridylate-rich cis-acting element (termed AU motif) (39) present in multiple copies in the 3`-UTRs of the transcripts that encode most cytokines and many early-response proteins. Because the mMCP transcripts do not possess an AU motif in their 3`-UTRs (Fig. 6), a different mechanism must control the steady-state levels of the varied chymase transcripts. The presence of the repetitive non-AU nucleotide sequence, ACCUACCUACCCAACUA, in 3`-UTRs of some Xenopus and Drosophila transcripts has been reported to influence their stability(40) . The mMCP-1, mMCP-2, and mMCP-4 transcripts all have multiple ``UGXCCCC'' sequences in their 3`-UTRs that are not present in the mMCP-5 transcript (Fig. 6). These non-AU repetitive sequences in the 3`-UTRs may be the cis-acting elements that regulate the steady-state levels of the mMCP-1, mMCP-2, and mMCP-4 transcripts.

Figure 6: Comparison of the 3`-UTRs of mast cell chymase transcripts. The nucleotide sequences of the 3`-UTRs of four mouse mast cell chymase mRNAs are depicted. The repetitive ``UGXCCCC'' sequence in mMCP-1(13, 27) , mMCP-2(6, 12) , and mMCP-4 (16) transcripts is underlined. The UAG and UAA translation stop codons are indicated in bold.

It has been established with use of recombinant inbred mouse strains and interspecific backcrosses that the genes that encode mMCP-1, mMCP-2, mMCP-4, mMCP-5, neutrophil cathepsin G, and four cytotoxic lymphocyte granzymes all reside at a complex on chromosome 14(6, 7, 8, 9) . Inasmuch as no crossover has been detected by restriction-enzyme fragment length polymorphism analysis of chromosomal DNA, either these 3-kb serine protease genes reside close to one another or some physical restraint in the particular region of chromosome 14 where they are located hinders recombination. Pulsed field gel electrophoresis of genomic DNA revealed that the mMCP-1, mMCP-2, mMCP-4, and mMCP-5 genes reside within 850 kb of one another(6) . Nucleotide sequencing studies revealed that another chymase gene, encoding mMCP-8, is located only 5-7 kb away from the mMCP-1 gene(41) . Recently, it was reported that a mouse T cell line (derived with IL-3 and IL-9 but maintained with IL-9 alone) expresses high steady-state levels of the transcripts that encode granzyme B, mMCP-1, mMCP-2, and mMCP-5(42) . Taken together, these findings raise the possibility that a common cis-acting enhancer element induces the transcription of many of the serine protease genes located at the chromosome 14 complex and that the steady-state level of each protease transcript is regulated by tissue-specific factors that instruct the cell which protease transcripts should be processed and/or protected from rapid degradation. Pertinent to this hypothesis is the fact that many genes that reside on chromosome 11 in the mouse (43, 44) encode cytokines whose expression is also post-transcriptionally regulated during inflammation.

Because the mast cell chymases are enzymatically active at neutral pH, it would be advantageous to limit their expression to a single cell type. Any cell could transcribe the mMCP-2 gene if it expresses the appropriate DNA binding protein that induces the transcription of the gene in mast cells or if a mutation was to occur in the promoter of the gene so that a new cis-acting motif is created that is recognized by a different transcription factor in that cell. Many hematopoietic effector cells (e.g. neutrophils, eosinophils, macrophages, natural killer cells, and cytotoxic lymphocytes) possess the chymase-zymogen processing enzyme dipeptidyl peptidase I (45) and therefore have the ability to convert any translated pro-mMCP-2 into mature, enzymatically active neutral protease. The post-transcriptional regulatory mechanism observed in the mast cell not only would accommodate cytokine regulation of protease expression during an inflammatory response but would also prevent the expression of the chymase family of mast cell proteases in other cell types if their genes were aberrantly transcribed.

FOOTNOTES

*

This work was supported by Grants AI-23483, AI-22531, AI-31599, AI-07306, AR-07530, AR-36308, and HL-36110 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Harvard Medical School, Seeley G. Mudd Bldg., 250 Longwood Ave., Boston, MA 02115. Tel.: 617-432-2838; Fax: 617-432-0979; rstevens{at}warren.med.harvard.edu.

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The abbreviations used are: mMCP, mouse mast cell protease; AU motif, adenylate- and uridylate-rich cis-acting element; bp, base pair; kb, kilobase pair(s); IL, interleukin; mBMMC, mouse bone marrow-derived mast cells; mMC-CPA, mouse mast cell carboxypeptidase A; PCR, polymerase chain reaction; RT, reverse transcription; UTR, untranslated region.

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The mMCP-1, mMCP-2, mMCP-4, and mMCP-5 gene-specific probes correspond to the 131-, 149-, 121-, and 200-bp 3`-UTRs of their respective cDNAs. The mMC-CPA gene-specific probe is 1.3 kb in size and corresponds to the full-length cDNA; the -actin gene-specific probe is 750 bp in size.

ACKNOWLEDGEMENTS

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