©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transforming Growth Factor Up-regulates Expression of the N-Acetylglucosaminyltransferase V Gene in Mouse Melanoma Cells (*)

(Received for publication, November 28, 1994; and in revised form, January 9, 1995)

Eiji Miyoshi (1) (2) Atsushi Nishikawa (1) Yoshito Ihara (1) Hiroyuki Saito (1) Naofumi Uozumi (1) Norio Hayashi (2) Hideyuki Fusamoto (2) Takenobu Kamada (2) Naoyuki Taniguchi (1)(§)

From the  (1)Department of Biochemistry and the (2)First Department of Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

beta-1,6-N-acetylglucosaminyltransferase V (GnT-V) (EC 2.4.1.155) that catalyzes beta-1,6 branching in asparagine-linked oligosaccharides is activated on viral or oncogenic transformation and is associated with tumor metastasis. To study the molecular mechanisms involved in regulation of expression of the GnT-V gene, we cloned cDNA and genomic DNA for the enzyme (Saito, H., Nishikawa, A., Gu, J., Ihara, Y., Soejima, Y., Sekiya, C., Niikawa, N., and Taniguchi, N.(1994) Biochem. Biophys. Res. Commun. 198, 318-327). We found that transforming growth factor beta (TGFbeta) specifically induced GnT-V expression in mouse melanoma cells. The activity of GnT-V was increased 24 h after the addition of TGFbeta and remained at high levels up to 72 h. Northern blot analysis showed that the mRNA levels of GnT-V were consistent with the increased activity. To further investigate the nature of the induction, mRNA stability and transcriptional activity were assayed. The enhancement of the GnT-V mRNA expression resulted from prolonged mRNA stability, not from increased transcription. Consequently, elevated mRNA levels were observed even 72 h after the addition of TGFbeta. Lectin blot analysis involving leukoagglutinin showed newly synthesized beta-1,6 branching structures in the sugar chains of a protein of approximately 130 kDa at 48 h after TGFbeta treatment. These results suggested that TGFbeta caused changes in the sugar chains of proteins in melanoma cells by up-regulating GnT-V expression.


INTRODUCTION

UDP-GlcNAc, alpha-mannoside beta-1,6-N-acetylglucosaminyltransferase V (GnT-V) (^1)yields beta-1,6-linked branching in complex-type, asparagine-linked oligosaccharides, as shown in Fig. S1(1, 2) . GnT-V is an interesting glycosyltransferase because some malignant phenotypes are associated with changes in its activity. For example, the transformation of baby hamster kidney cells caused by a tumor virus results in increases in GnT-V activity and alteration of cell-surface glycosylation(3, 4) . Recently, GnT-V was purified from rat kidney (5) and human lung cancer cells(6) , and cloning of its cDNA and chromosomal mapping were achieved(7, 8) . We reported (9) that expression of GnT-V was induced in the early stages of hepatocarcinogenesis in rodent models and that the increased levels of GnT-V activity were consistent with the increased mRNA levels.


Scheme 1: Scheme 1.



Transforming growth factor beta1 (TGFbeta) is widely referred to as a prototypical epithelial cell inhibitor(10, 11) . Although it inhibits the growth of both normal melanocytes and melanoma cells(12, 13) , TGFbeta is expressed at higher levels in many cancers, including melanomas, than in normal tissue(14, 15, 16) . TGFbeta also exhibits a number of properties that can promote tumor growth. It has been found to stimulate angiogenesis(10) , to suppress the immune system(17, 18) , and to modulate cellular adhesion and migration on an extracellular matrix(19, 20) . The expression of TGFbeta by cancer cells is thought to be related to their malignant phenotype(20, 21) .

The structures of sugar chains have a lot of biological functions, including cell differentiation, cell adhesion, viral infection, and the action of hormones(22) . Dennis et al.(23, 24) reported that high GnT-V activity and beta-1,6 branching structures of sugar chains were directly associated with metastasis. The molecular mechanism by which the GnT-V activity increases is, however, unknown. In this study, we found that the expression of GnT-V was induced specifically by TGFbeta in some cytokines and found that beta-1,6 branching structures in the sugar chains of proteins increased after TGFbeta treatment. The mechanism underlying the enhancement of GnT-V was investigated at the RNA level.


MATERIALS AND METHODS

Cell Culture

Mouse B16 melanoma cells, F1 and F10(25) , were kindly provided by Dr. Shun'ichirou Taniguchi (Dept. of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University Medical School). About 10^5 cells were cultured in RPMI 1640 medium (Nakarai Tesque, Japan) containing 10% fetal bovine serum (Life Technologies, Inc.), 50 units/ml penicillin, and 100 µg/ml kanamycin at 37 °C. After 48 h, the medium was changed to serum-free RPMI 1640 medium, and various concentrations of TGFbeta (Nakarai Tesque, Japan), interleukin 1beta and interleukin 2 (Otsuka Pharmaceutical Co., Japan), and hepatocyte growth factor (kind gift from Dr. T. Nakamura, Biomedical Research Center, Osaka University Medical School) were added. Each experiment started at this time. The morphological changes after TGFbeta treatment were observed and photographed with a microscope camera (PM-10AD, Olympus, Japan). After harvesting with phosphate-buffered saline containing 0.5 mM EDTA, the cells were stained with 0.3% trypan blue and then counted on a turk tube through a light microscope.

Assay of Glycosyltransferases

Sonicated cells were subjected to enzyme assay for glycosyltransferases. The cells were washed with phosphate-buffered saline twice and then centrifuged at 1500 times g for 10 min. The precipitated cells were resuspended in 100-200 µl of phosphate-buffered saline and then sonicated. The activities of beta-1,4-N-acetylglucosaminyltransferase III, GnT-V, and beta-1,4-galactosyltransferase were assayed using a fluorescence-labeled, pyridylaminated biantennary sugar chain as the substrate(26) . The details of the standard assay were previously given (27, 28) .

Northern Blot Hybridization and Stability Assay for GnT-V mRNA

Total RNA was prepared from the cells according to Chomczynski and Sacchi(29) . Northern blot hybridization was performed as previously described(9) . To determine the relative stability of GnT-V mRNA, F1 cells were incubated in serum-free medium with or without 5 ng/ml TGFbeta for 48 h, and then transcription of the endogenous gene was inhibited by the addition of 100 µM 5,6-dichlorobenzimidazole riboside (DRB) (Sigma). RNA was extracted from the cells after different incubation times up to 18 h and then analyzed by Northern blot hybridization. The intensities of the labeled bands were measured with a densitometer (CS-9000, Shimadzu, Japan).

Determination of Transcriptional Activity

Nuclei were isolated from 8 times 10^6 F1 cells that had been incubated in the presence or absence of 5 ng/ml TGFbeta for 0-72 h. A nucleolar transcription assay was performed as previously described (30, 31) with minor modifications. Briefly, collected cells were suspended in Nonidet P-40 lysis buffer (10 mM Tris-HCl, pH 7.4, containing 10 mM NaCl, 3 mM MgCl(2), and 0.5% Nonidet P-40), incubated on ice, and then centrifuged at 1,500 rpm for 5 min. The precipitated nuclei were suspended in nuclei storage buffer (10 mM Tris-HCl, pH 8.0, containing 40% glycerol, 5 mM MgCl(2), and 0.1 mM EDTA), quickly frozen in liquid nitrogen, and then stored in the same buffer until used. Approximately 3 times 10^6 nuclei were rapidly thawed and added to the reaction mixture containing 1 mM each of ATP, CTP, and GTP, 5 mM dithiothreitol, 300 mM KCl, 5 mM MgCl(2), and 150 µCi of [P]UTP. The labeling reaction was performed at 30 °C for 1 h with occasional shaking. The reaction was terminated by the addition of 9 volumes of nuclear digestion buffer(32) , followed by incubation with proteinase K (Sigma) for 1 h at 60 °C. Extraction and purification of the P-labeled nascent RNA was performed as described elsewhere(33) . An equal amount of radiolabeled RNA (3 times 10^6 cpm) was added to each membrane, and hybridization was carried out as for slot-blot analysis for 48 h at 42 °C.

Nylon membranes (Zeta-Probe, Bio-Rad) were prepared with immobilized GnT-V cDNA as described below. Bluescript II KS (Stratagene) containing GnT-V cDNA (9) was linearized with restriction enzymes and then precipitated with ethanol. The linearized plasmid was denatured in 0.5 M NaOH and 2 M NaCl for 1 h at room temperature, neutralized with 6 times SSC, and then immediately blotted onto a nylon membrane using a slot-blot apparatus (filtration manifold system, series 1055, Life Technologies, Inc.). The membrane was hybridized with the radiolabeled RNA and then washed as described for Northern blot hybridization. After development, the intensities of the labeled bands were measured with a densitometer (CS-9000, Shimadzu, Japan).

Lectin Blot Analysis

3 or 10 µg of proteins from homogenized F1 cells were electrophoresed on a 12% SDS-acrylamide gel. After SDS-polyacrylamide gel electrophoresis, one gel (10 µg of proteins) was stained with Coomassie Brilliant Blue R-250. Another gel was blotted onto a nitrocellulose filter with instructions TE70 and TE77 Semiphor Semi-dry transfer units (Hoefar Scientific Instruments, San Francisco, CA). The filter was prehybridized with 3% bovine serum albumin for 3 h and then incubated with 2 µg/ml biotin-L-PHA (Seikagaku Industrial Corp., Tokyo) overnight. After washing for 10 min (4 times with Tris-buffered saline containing 0.05% Tween 20), it was incubated with 1/2000 times diluted horseradish peroxidase avidin D (Vector Industrial Corp., CA) for 2 h and then washed for 10 min (4 times with Tris-buffered saline containing 0.05% Tween 20). Staining was performed with Western blot detection reagents (ECL, Amersham Corp.).


RESULTS

Expression of GnT-V mRNA after Incubation with Various Reagents

Since it was known that GnT-V is induced in various cells including transformed, metastatic and hepatoma cells, we investigated the effects of various agents related to cell growth or transformation on the expression of GnT-V. After 48 h of incubation with various reagents, the level of GnT-V mRNA was increased only by TGFbeta treatment in both F1 and F10 cells (Fig. 1). Interleukin 1beta, interleukin 2, and hepatocyte growth factor treatment did not affect GnT-V mRNA. TGFbeta was found to be a strong inducer of GnT-V. The mechanism by which TGFbeta acts on GnT-V expression was investigated as follows.


Figure 1: Expression of GnT-V mRNA after incubation with various reagents. F1 and F10 cells were cultured in the presence of interleukin (IL) 1beta (5 ng/ml), interleukin 2 (250 units/ml), TGFbeta (5 ng/ml), or hepatocyte growth factor (5 ng/ml) or in the absence of these reagents (control). After 48 h of incubation with these reagents, total RNAs were extracted, and Northern blot analysis was performed as described under ``Materials and Methods.'' HGF, hepatocyte growth factor.



Effects of TGFbeta on Cell Growth and Morphology

To investigate the effects of TGFbeta on cell growth, cells were counted at the indicated times after the addition of TGFbeta (Fig. 2). TGFbeta, a prototypic epithelial cell inhibitor, also inhibited the growth of F1 cells by 18-33% under serum-free conditions. The cells grew to a confluent state after 48-72 h of incubation in the presence or absence of TGFbeta. Morphological changes were found after 24-48 h of TGFbeta treatment (Fig. 3). Both F1 and F10 cells were originally spindle shaped. After treatment with TGFbeta, the cells had longer processes than the untreated controls. Furthermore, even though the cell numbers were decreased, the gaps between the cells were found to be narrow.


Figure 2: Changes in cell count after TGFbeta treatment. The number of cells was counted after incubation with (bullet) or without (circle) TGFbeta (5 ng/ml) for the times indicated. Each point is the average of three values. Each bar indicates the standard deviation at the point.




Figure 3: Morphological changes in F1 and F10 cells after TGFbeta treatment. F1 and F10 cells were cultured in the presence or absence of TGFbeta (5 ng/ml) for 24 h and then photographed (original magnification, 200times).



Changes in GnT-V Activity after TGFbeta Treatment

The activity of GnT-V was assayed at various times after the addition of TGFbeta (Fig. 4). The activity gradually increased after 24 h of incubation with TGFbeta, while it did not change in untreated F1 cells. The increase in activity was apparent after the morphological changes appeared. The activities of beta-1,4-N-acetylglucosaminyltransferase III and beta-1,4-galactosyltransferase were assayed at this time (Table 1). beta-1,4-N-Acetylglucosaminyltransferase III activity was below the detection level in F1 cells and was not induced by TGFbeta. beta-1,4-galactosyltransferase activity did not change after TGFbeta treatment. Induction by TGFbeta was restricted GnT-V among these glycosyltransferases.


Figure 4: Effect of TGFbeta on GnT-V activity in F1 cells. Cells were cultured in the presence (bullet) or absence (circle) of TGFbeta (5 ng/ml). GnT-V activity was assayed as described under ``Materials and Methods.'' Each point is the average for triplicate experiments. Each bar indicates the standard deviation at the point.





The effects of various concentrations of TGFbeta on GnT-V activity were investigated in both F1 and F10 cells (Fig. 5). GnT-V activity increased in proportion to the concentration of TGFbeta up to 2 ng/ml for F10 cells and 5 ng/ml for F1 cells. The activity in F10 cells decreased at TGFbeta concentrations of more than 5 ng/ml. In F10 cells, GnT-V activity was induced at lower concentrations of TGFbeta than in F1 cells, although the initial levels were not different.


Figure 5: Changes in GnT-V activity in F1 and F10 cells after incubation with various concentrations of TGFbeta. F1 (circle) and F10 () cells were cultured in the presence of various concentrations of TGFbeta for 48 h. GnT-V activity was assayed as described under ``Materials and Methods.'' Each point is the average for triplicate experiments. Each bar indicates the standard deviation at the point.



Expression of GnT-V mRNA on TGFbeta Treatment

To further investigate the nature of the induction described above, Northern blot analysis was performed. The response of the GnT-V mRNA level to TGFbeta stimulation was found to be time dependent (Fig. 6). GnT-V mRNA was detected as a major band corresponding to about 7 kilobases and a minor band to about 3.5 kilobases. Both of these bands were induced by 5 ng/ml TGFbeta. The enhancement of GnT-V mRNA expression was remarkable after 24 h of incubation with TGFbeta and continued until 72 h. These increases corresponded well with the levels of the enzyme activity. No changes in the mRNA level were observed in untreated cells.


Figure 6: Effect of TGFbeta on expression of GnT-V mRNA. F1 cells were cultured in the absence (A, C, E) or presence (B, D, F) of TGFbeta (5 ng/ml). Total RNA (30 µg) extracted from the cells at the indicated times was electrophoresed on a 1.0% agarose gel containing 2.2 M formaldehyde. The membrane filter was hybridized with P-labeled GnT-V cDNA (A, B) or beta-actin (C, D). Ethidium bromide staining shows a comparable amount of RNA in each lane (E and F).



Effect of TGFbeta on GnT-V mRNA Stability

The induction of GnT-V activity by TGFbeta was dependent on enhanced mRNA expression. This effect, however, appeared relatively late after the addition of TGFbeta, which suggested a secondary effect or a change in mRNA stability. To determine whether or not TGFbeta increased the stability of GnT-V mRNA, we blocked transcription of the gene by the addition of DRB and then studied the decay of the mRNA with time (Fig. 7). As expected, the mRNA level was highest in TGFbeta-treated F1 cells right after DRB blocking, and then disappearance of the GnT-V mRNA bands was observed. In control cultures, GnT-V mRNA decayed more rapidly than in TGFbeta-treated cultures and was scarcely detected after 7.5 h of DRB treatment. The half-life of GnT-V mRNA, as measured by densitometry, increased to about 2.3-fold in the TGFbeta-treated cells (data not shown).


Figure 7: Effect of TGFbeta on the stability of GnT-V mRNA. F1 cells were cultured in the presence or absence of TGFbeta (5 ng/ml) for 48 h. Total RNA was isolated from one set of control and TGFbeta-treated cultures and used to determine the GnT-V mRNA level at t = 0. The remaining cultures were placed in fresh medium containing 100 µM DRB with or without TGFbeta (5 ng/ml), followed by incubation for an additional 3, 4.5, 7.5, 12, or 18 h. Total RNA (30 µg) was isolated and analyzed for GnT-V mRNA by Northern blot hybridization.



Effect of TGFbeta on Transcriptional Activity

Nuclear run-off experiments were performed to determine whether or not the elevation of the mRNA level was the result of increased transcription mediated by TGFbeta. As shown in Fig. 8A, no increase in the transcriptional level of the GnT-V gene was observed in nuclei isolated from cells treated with TGFbeta, although the transcriptional level of the beta-actin gene was changed a little. Fig. 8B shows typical results of the transcriptional assay with dot-blot analysis. The transcriptional activity of GnT-V was not changed at 24 h after TGFbeta treatment. Therefore, the enhanced expression of GnT-V mRNA was not the result of transcriptional activation but of increased stability of the mRNA.


Figure 8: Effect of TGFbeta on transcription of the GnT-V gene in nuclei from F1 cells. Nuclei were isolated from control cells and cells treated with TGFbeta for 0-72 h. After labeling with [P]UTP, nascent RNA was purified and hybridized to slots containing various concentrations of immobilized Bluescript DNA containing GnT-V cDNA. beta-Actin and Bluescript (BS) DNA were used as positive and negative control, respectively. The details of the assay procedure are given under ``Materials and Methods.'' The relative intensities of the labeled bands, GnT-V (circle) and beta-actin (box) transcriptional levels in control F1 cells, and GnT-V (bullet) and beta-actin () transcriptional levels in TGFbeta-treated cells were measured, respectively, with a densitometer (A). Data indicate the results for 1-3 bands. Representative results of these experiments are shown in B.



Lectin Blot Analysis

L-PHA binds to the beta-1,6 branches in asparagine-linked oligosaccharides, which are the products of GnT-V(34) . To determine whether or not the structures of sugar chains were changed by the increase in GnT-V activity, L-PHA blot analysis was performed. There were no differences in total cellular proteins except lane6, as judged on SDS-polyacrylamide gel electrophoresis, between control and TGFbeta-treated F1 cells (Fig. 9A). However, a band corresponding to approximately 130 kDa, indicated by an arrow, was detected for TGFbeta-treated F1 cells on L-PHA blot analysis (Fig. 9B). This band was very faint for control cells, while a band of approximately 80 kDa was observed for both control and TGFbeta-treated F1 cells. As shown in Fig. 2, cell numbers were decreased after 72 h of incubation with TGFbeta, and much more decreased after 96 h (data not shown). Cell viability was not good at this point. As shown in Fig. 9B, both the 130-kDa band and the 80-kDa band were decreased at 96 h after TGFbeta treatment.


Figure 9: SDS-polyacrylamide gel electrophoresis analysis and lectin blot analysis of F1 cells treated with TGFbeta. A, 10 µg of total cellular proteins from homogenized F1 cells at 48 h (lane1), 72 h (lane2), and 96 h (lane3) without any treatment or at 48 h (lane4), 72 h (lane5), and 96 h (lane6) after TGFbeta treatment were electrophoresed on a 12% agarose gel and then stained. B, 3 µg of the total cellular proteins mentioned above were blotted onto a nitrocellulose membrane, and then staining with L-PHA was performed as described under ``Materials and Methods.'' The numbers at the left indicate the molecular weights of standards.




DISCUSSION

TGFbeta is a pleotrophic peptide that was originally shown to induce a transformed phenotype in normal fibroblasts cultured in soft agar(35) . It also regulates the growth of certain cell types, primarily by inhibiting cell proliferation(12, 13, 14, 15) . Although the growth of F1 cells was inhibited by 18-33% on incubation with TGFbeta (Fig. 2), GnT-V activity was up-regulated by the TGFbeta treatment (Fig. 4). Therefore, the effect of TGFbeta on the expression of GnT-V appears to be separate from the function of the growth factor as a prototypical epithelial cell inhibitor. Hahn and Goochee (36) reported that GnT-V activity was higher in proliferating states and decreased in confluent cultures. For this reason, the GnT-V activity in control F1 cells slightly increased with time.

Melanoma cells differ from normal melanocytes in morphology, life span, and chromosomal abnormality. Morphology also differs between primary melanoma and metastatic phenotypes(37) . Heffernan et al.(38) reported that beta-all-trans-retinoic acid-induced endodermal differentiation of mouse teratocarcinoma cells, F9, was accompanied by changes in the expression of beta-1,6-N-acetylglucosamine transferase and polylactosamine. Abrupt induction of GnT-V activity was observed in F9 cells after 4 days of retinoic acid treatment. Morphological changes accompanying cell differentiation appeared at this time. While the GnT-V activity in F1 cells gradually increased after incubation with TGFbeta and other glycosyltransferases did not change ( Fig. 4and Table 1), suggesting that different induction mechanisms are triggered by TGFbeta and retinoic acid.

Melanoma cells produce a variety of growth factors, including basic fibroblast growth factor, platelet-derived growth factor, transforming growth factors alpha and beta (TGFalpha and TGFbeta), interleukins 1alpha and 1beta, and melanoma growth stimulatory activity(39) . The F1 and F10 cells, however, exhibited different sensitivities to exogenous TGFbeta (Fig. 5). The GnT-V activity in F10 cells increased at lower concentrations of TGFbeta than that in F1 cells did. This might reflect the high metastatic character of F10 cells. However, GnT-V activities in steady states and L-PHA blotting pattern between F1 and F10 cells were almost the same. The 130-kDa band as shown in Fig. 9B was induced by more than 2 ng/ml TGFbeta in both cells (data not shown). No other differences except sensitivities to exogenous TGFbeta were observed between F1 and F10 cells in the present experiments.

TGFbeta increased GnT-V activity by enhancing mRNA expression (Fig. 6). The level of GnT-V mRNA increased after 24 h of incubation with TGFbeta and remained high up to 72 h. Because direct induction of GnT-V gene expression would likely follow a shorter time course, a secondary effect of TGFbeta on GnT-V expression was suggested. One possible mechanism involves increasing stability of the mRNA. There is some evidence that up-regulation of extracellular matrix synthesis by TGFbeta also affects post-transcriptional steps. For example, TGFbeta treatment has been shown to increase the half-lives of mRNAs for alpha1(I) collagen in confluent 3T3 fibroblasts(40) , heparan sulfate proteoglycan in colon cancer cells(41) , and elastin in human skin fibroblasts(42) . In contrast, up-regulation of collagen type II by TGFbeta in rabbit articular chondrocytes (43) and glucocorticoids in normal human T lymphocytes (44) involves increased transcription of the genes without apparent changes in mRNA stability. TGFbeta appears to up-regulate the GnT-V gene through the former mechanism ( Fig. 7and 8). The half-life of GnT-V mRNA increased approximately 2.3-fold after TGFbeta treatment. The persistence of an elevated level of GnT-V mRNA even after 72 h incubation with TGFbeta (Fig. 6) is in keeping with this mechanism. Although high activities of GnT-V are observed in transformation and carcinogenesis(3, 4, 9) , the mechanism underlying the high expression remains to be elucidated. The induction of GnT-V by TGFbeta is one possible pathway. The enhancement of GnT-V expression by TGFbeta resembled that of some extracellular matrix proteins induced by TGFbeta(40, 41, 42) . Such regulation might be of significance as to characteristics of GnT-V, a glycosyltransferase associated with tumor metastasis.


FOOTNOTES

*
This work was supported in part by grants-in-aid for cancer research and scientific research on priority areas from the Ministry of Education, Science, and Culture of Japan. 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 all correspondence and reprint requests should be addressed. Fax: 81-6-879-3429.

(^1)
The abbreviations used are: GnT-V, beta-1,6-N-acetylglucosaminyltransferase V; TGFbeta, transforming growth factor beta; DRB, 5,6-dichlorobenzimidazole riboside; L-PHA, leukoagglutinin.


ACKNOWLEDGEMENTS

We thank Prof. Shun'ichirou Taniguchi (Dept. of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University Medical School) for giving us the F1 and F10 cells, Mari Tabata for assistance in culturing the cells, and Stephanie House for editing this manuscript.


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