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Inhibition of p38 Mitogen-Activated Protein Kinase Enhances Adrenergic-Stimulated Arylalkylamine N-Acetyltransferase Activity in Rat Pinealocytes

Departments of Physiology and Medicine (C.L.C.) Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Address all correspondence and requests for reprints to: Dr. A. K. Ho, Department of Physiology, 7-26 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. E-mail: anho{at}ualberta.ca.

	Abstract

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We have previously shown that inhibition of p38^MAPK increases adrenergic-stimulated p42/44^MAPK activation in rat pinealocytes. In this study we investigated whether p38^MAPK played a role in the adrenergic regulation of arylalkylamine-N-acetyltransferase (AA-NAT) induction and melatonin (MT) synthesis. Treatment of pinealocytes with norepinephrine (NE) caused a time-dependent increase in the levels of AA-NAT mRNA, AA-NAT protein, and enzymatic activity as well as MT production. Cotreatment with SB202190, a selective p38^MAPK inhibitor, although having no effect on AA-NAT activity or protein level 3 h after NE treatment, caused a sustained increase in AA-NAT activity and protein level after 6 h of NE treatment. The increases in NE-stimulated AA-NAT activity and protein level by SB202190 occurred in the absence of an increase in AA-NAT mRNA. Similar results were obtained when AA-NAT was induced by (Bu)₂cAMP or when SB203580 was used to inhibit p38^MAPK. In comparison, SB202474, the inactive analog, had no effect on NE or (Bu)₂cAMP-stimulated AA-NAT activity or protein level. SB202190 also increased cumulative NE-stimulated MT production, provided that the medium was supplemented with 5-methoxytryptamine. p38^MAPK inhibitors had no effect on hydroxyindole-O>-methyltransferase activity. These results show that inhibition of p38^MAPK, although having no effect on cAMP-mediated AA-NAT transcription, appears to increase AA-NAT activity either by increasing translation or by reducing degradation of the AA-NAT protein. The lack of effect on NE-stimulated MT accumulation by p38^MAPK inhibitors in the absence of 5-methoxytryptamine could be secondary to a lack of substrate, or alternatively, hydroxyindole-O-methyltransferase may become limiting.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE RAT PINEAL gland is stimulated by the nightly release of norepinephrine (NE) from the sympathetic nerve terminals (1). NE, by activating both - and ß-adrenergic receptors, causes 100-fold increases in cAMP and cGMP accumulation (1). The primary function of cAMP in the rat pineal gland is to initiate a series of cellular processes that lead to the synthesis of arylalkylamine-N-acetyltransferase (AA-NAT), the rate-limiting enzyme in synthesis of the pineal hormone, melatonin (MT) (2). These cellular events include activation of cAMP-dependent protein kinase and phosphorylation of the transcription factor cAMP response element-binding protein (CREB), followed by an up to 150-fold increase in transcription of AA-NAT mRNA (2). At the posttranslational level, cAMP also plays an important role in maintaining AA-NAT activity by switching the fate of AA-NAT protein from destruction by proteasomal proteolysis (3) to protection and activation (4). Increased AA-NAT activity results in an elevation of the intracellular concentration of N-acetylserotonin, which is converted to MT by hydroxyindole-O-methyltransferase (HIOMT) (5). In contrast to cAMP, the physiological importance of NE-stimulated cGMP accumulation in the pineal gland is not as well established. However, we have shown that the NEcGMP pathway is the main signaling mechanism involved in activation of p42/44^MAPK (6, 7, 8), a member of the MAPK family of kinases.

MAPKs, a large family of serine/threonine protein kinases, have important functions as mediators of signal transduction and are activated by diverse stimuli, such as cytokines, growth factors, neurotransmitters, hormones, and cellular stresses (9, 10). Three main groups of MAPKs have been identified: p42/44^MAPK (11, 12), p38^MAPK (13, 14), and c-Jun N-terminal kinase (15, 16). In rat pinealocytes, we have demonstrated activation of p42/44^MAPK (6, 7) and its downstream kinase, the 90-kDa ribosomal S6 kinase by NE (8). Interestingly, we also found that NE activation of p42/44^MAPK can be modulated by the state of p38^MAPK activation (17). Using specific p38^MAPK inhibitors, we showed that inhibition of p38^MAPK amplifies adrenergic-stimulated p42/44^MAPK phosphorylation (17). This finding is in agreement with the mounting evidence that cross-talk between the p38^MAPK pathway and other members of the MAPK family represents an important mechanism of biological response regulation (18, 19, 20).

The observation that inhibition of p38^MAPK can significantly affect the adrenergic-stimulated phosphorylation of p42/44^MAPK suggests that it may also modulate other adrenergic-mediated events, such as AA-NAT induction. Moreover, because p42/44^MAPK mediates its cellular responses through phosphorylation of nuclear transcription factors such as CREB (21, 22, 23), and CREB phosphorylation is important in the circadian activation of AA-NAT (24), p38^MAPK may affect MT biosynthesis in the pineal gland. To explore this possibility, we investigated the effect of p38^MAPK inhibition on basal and stimulated AA-NAT induction and MT production.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
(Bu)₂cAMP, NE, tryptamine hydrochloride, and antibodies against p38^MAPK and phosphorylated p38^MAPK were obtained from Sigma-Aldrich Corp. (St. Louis, MO). SB202190, SB 203580, and SB202474 were obtained from Calbiochem (San Diego, CA). [³H]Acetyl-coenzyme A was purchased from NEN Life Science Products (Boston, MA). [³H]MT (specific activity, 81.1 Ci/mmol) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Polyclonal antibodies for the RIA of MT were obtained from CIDTech Co. (Mississauga, Canada). Antibody against AA-NAT (AB3314) was a gift from Dr. D. C. Klein (NICHHD, NIH, Bethesda, MD). All other chemicals were of the purest grade available commercially.

Preparation of pinealocytes
All procedures related to animal usage were reviewed and approved by the health sciences animal and welfare committee of University of Alberta. Sprague Dawley rats (male; weighing 150 g) were obtained from the University of Alberta animal unit. Pinealocytes were prepared by trypsinization from freshly dissected rat pineal glands as described previously (25). Cells were suspended in DMEM containing 10% fetal calf serum and were maintained at 37 C for 24 h in a gas mixture of 95% air and 5% CO₂ before experiments. Aliquots of pinealocytes (5 x 10⁵ cells/0.5 ml) were treated with drugs that had been prepared in concentrated solutions in water or dimethylsulfoxide for the duration indicated. Treated cells were collected by centrifugation (2 min, 12,000 x g). Pinealocyte total RNAs were isolated and purified by RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instruction. Samples for Western blot analysis were solubilized in 1x sample buffer by boiling for 5 min and were stored until electrophoresis. The homogenization buffer contained 20 mM Tris-HCl; 2 mM EDTA; 0.5 mM EGTA; 2 mM phenylmethylsulfonylfluoride; 1 µg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM sodium orthovanadate; and 1 mM sodium fluoride (pH 7.5). Samples for determination of NAT and HIOMT activities were immediately frozen in dry ice and stored at -75 C. Media were collected for MT determination.

RT-PCR analysis
First strand cDNA was synthesized using 1 µg total RNA, which had been treated with ribonucleaase-free deoxyribonuclease, by using the Superscript preamplification system (Life Technologies, Inc., Grand Island, NY) and oligo(deoxythymidine) primer according to the manufacturer’s instructions. After first strand cDNA synthesis, PCR was performed in a 50-µl reaction mixture containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 100 µM of each deoxy-NTP, 1.25 U Taq polymerase (PerkinElmer Cetus, Emeryville, CA), and 50 pmol of a previously characterized primer pair for AA-NAT (26) (forward, TGTCTTGTGGCCTTCATCATCGG; reverse, TGCGCAGCTTGGGTCAGCAGCC). After denaturing for 3 min at 94 C, PCR was performed for 30 cycles (1 min at 94 C, 1 min at 63 C, and 1 min at 72 C). A final extension was carried out at 72 C for 5 min. Reference cDNA was generated from extracts of NE-treated pinealocytes incubated along with the experimental samples. Initial cDNA concentrations were confirmed by PCR reactions amplifying cytoplasmic ß-actin as an internal standard (not shown). All reaction sets included water blanks as negative controls. The amplified products were separated on 1.5% agarose gels, and images were analyzed using a Kodak 2000R imaging station with Kodak 1-D software (Eastman Kodak Co., Rochester, NY).

Western blot
SDS-PAGE was performed according to the procedure of Laemmli (27) using 10% acrylamide (Mini-Protein II gel system, Bio-Rad Laboratories, Hercules, CA). After electrophoresis, gels were equilibrated for 20 min in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). Proteins were transferred onto polyvinylidene difluoride membranes (1 h, 100 V), which were incubated with a blocking solution [5% dried skim milk in 100 mM Tris (pH 7.5) with 140 mM NaCl and 0.01% Tween 20] for a minimum of 1.5 h. The blots were then incubated overnight at 4 C with diluted specific antisera as indicated. After washing three times with blocking solution, blots were incubated with diluted with horseradish peroxidase-conjugated second antibodies (Bio-Rad Laboratories) for 1 h at room temperature. They were then washed extensively and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech).

Enzymatic assays
AA-NAT activity was determined as described previously (28). Briefly, treated pinealocytes were stored frozen in dry ice until homogenization in a reaction mixture of 0.1 M phosphate buffer (pH 6.8) containing 30 nmol [³H]acetyl coenzyme A (specific activity, 1 mCi/mmol) and 1 µmol tryptamine hydrochloride in a final volume of 60 µl. The reaction mixture was incubated at 37 C for 1 h. At the end of the incubation period, the reaction was stopped by the addition of 1 ml methylene chloride. After vortexing, the aqueous phase was removed, and the organic phase was washed three times with 0.1 M phosphate buffer (pH 6.8). The organic phase was transferred to a scintillation vial and evaporated to dryness, and radioactive acetylated product was determined by scintillation counting. AA-NAT activity was expressed as nanomoles per hour per 10⁵ cells. HIOMT activity was determined using a previously established method (29) similar to the AA-NAT assay. The reaction mixture used for the HIOMT assay was a 0.1 M phosphate buffer (pH 7.9) that contained 1 mM [³H]S-adenosyl-L-methionine (specific activity, 5 mCi/mmol) and 1 mM N-acetylserotonin. The incubation conditions and product extraction are described above.

MT assay
Briefly, medium MT was extracted from 300 µl medium by vortexing with 1 ml methylene chloride. As 5-methoxytryptamine (0.1 mM) was added to the medium in selected experiments, washing the methylene chloride extracted medium five times with 0.25 ml 0.1 M HCl was included in the procedure. This washing effectively removed any interference of 5-methoxytryptamine in the MT RIA. After centrifugation, the organic phase was collected and evaporated to dryness. The residue was reconstituted in 500 µl assay buffer (0.01 M phosphate buffer, pH 6.5, containing 0.1% gelatin). The recovery of medium MT was greater than 98%. The extracted MT was assayed by RIA as described previously (29).

Statistical analysis
For the Western blots and RT-PCR results, a typical image from at least three similar experiments was presented. Results from the enzymatic assays and MT measurements were analyzed by paired t test or ANOVA with Duncan’s multiple range test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of p38^MAPK inhibitors on NE-stimulated AA-NAT activity
The effect of SB202190 (10 µM), a specific p38^MAPK inhibitor (30, 31), on the time profile of NE-stimulated AA-NAT activity is shown in Fig. 1A. Treatment with NE (1 µM) caused a significant increase in AA-NAT activity at 3 h (P < 0.05) that peaked at 6 h. The increase in AA-NAT activity was sustained for at least another 6 h and was followed by a gradual decline. SB202190 (10 µM) alone had no effect on AA-NAT activity. However, whereas the presence of SB202190 (10 µM) had no effect on NE-stimulated AA-NAT activity when measured 3 h after NE stimulation, it significantly enhanced (up to 70%) the NE-stimulated responses at later time points (P < 0.05 between 6 and 21 h after NE stimulation). A concentration-response study indicated that treatment with SB202190 caused an increase in NE-induced maximal AA-NAT activity (Fig. 1B). SB203580 (10 µM), another active p38^MAPK inhibitor, had a similar enhancing effect on NE-stimulated AA-NAT activity 12 h after drug addition (Table 1). In comparison, SB202474 (10 µM), the negative control, had no effect on NE-stimulated AA-NAT activity 12 h after drug addition (Table 1).

View larger version (23K): [in this window] [in a new window]   FIG. 1. Effect of a p38MAPK inhibitor on NE-stimulated AA-NAT activity. Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated with NE (1 µM) for different time periods (A) or different concentrations of NE for 12 h (B) in the presence or absence of SB202190 (10 µM) as indicated. The cells were then collected by centrifugation and assayed for AA-NAT activity as described in Materials and Methods. Each value represents the mean ± SEM of determinations from three independent experiments.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Effect of a p38MAPK inhibitor on (Bu)2cAMP-stimulated AA-NAT activity. Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated with (Bu)2cAMP (1 mM) for different time periods (A) or different concentrations of (Bu)2cAMP for 12 h (B) in the presence or absence of SB202190 (10 µM) as indicated. The cells were then collected by centrifugation and assayed for AA-NAT activity as described in Materials and Methods. Each value represents the mean ± SEM of determinations from three independent experiments.

View larger version (51K): [in this window] [in a new window]   FIG. 3. Effect of p38MAPK inhibitors on NE-stimulated AA-NAT mRNA. Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated with NE (1 µM) for different time periods in the presence or absence of 10 µM SB 202190 or SB202474 as indicated. The cells were then collected by centrifugation, total RNA was isolated, and cDNA was prepared. RT-PCR was performed using AA-NAT primers. The gel presented is representative of three separate experiments. Reference cDNA samples for each time point were prepared from pooled NE-treated pinealocyte extracts. See Materials and Methods for details.

View larger version (53K): [in this window] [in a new window]   FIG. 4. Effects of p38MAPK inhibitors on NE-stimulated AA-NAT protein. Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated with NE (1 µM) for different time periods in the presence or absence of 10 µM SB202190, SB202474, or SB203580 as indicated. The cells were then collected by centrifugation, dissolved in 1x sample buffer, and analyzed by Western blotting using a specific antibody against AA-NAT protein as described in Materials and Methods. The blots presented are representative of three separate experiments.

View larger version (21K): [in this window] [in a new window]   FIG. 5. Effect of a p38MAPK inhibitor on the dose response of NE-stimulated MT accumulation. Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated with different concentrations of NE for 12 h in the presence or absence of SB202190 (10 µM) as indicated in control medium (A) or medium supplemented with 0.1 mM 5-methoxytryptamine (B). After centrifugation, the medium was extracted with methylene chloride before MT determination by RIA as described in Materials and Methods. Each value represents the mean ± SEM of determinations from three independent experiments.

View larger version (21K): [in this window] [in a new window]   FIG. 6. Effect of a p38MAPK inhibitor on the dose response of NE-stimulated AA-NAT activity. Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated with different concentrations of NE for 12 h in the presence or absence of SB202190 (10 µM) as indicated in control medium (A) or medium supplemented with 0.1 mM 5-methoxytryptamine (B). The cells were then collected by centrifugation and assayed for AA-NAT activity as described in Materials and Methods. Each value represents the mean ± SEM of determinations from three independent experiments.

View larger version (29K): [in this window] [in a new window]   FIG. 7. Effect of a p38MAPK inhibitor on the time-course of NE-stimulated MT accumulation. Pinealocytes (5 x 104 cells/0.5 ml) were cultured for 24 h and treated with NE (1 µM) for different time periods in the presence or absence of SB202190 (10 µM) as indicated in control medium (A) and medium supplemented with 0.1 mM 5-methoxytryptamine (B). After centrifugation, the medium was extracted with methylene chloride before MT determination by RIA as described in Materials and Methods. Each value represents the mean ± SEM of determinations from three independent experiments.

View larger version (41K): [in this window] [in a new window]   FIG. 8. Effect of p38MAPK inhibitors on HIOMT activity. In the in vitro experiment, 3 x 106 pinealocytes were collected by centrifugation and homogenized in HIOMT assay buffer. An aliquot of the homogenate was used for HIOMT activity determination in the absence or presence of 10 µM SB202190, SB203580, or SB202474 in the reaction mixture as indicated. In the in vivo experiment, pinealocytes were cultured for 24 h and treated with 10 µM p38MAPK inhibitor for 12 h as indicated. The cells were then collected by centrifugation and assayed for HIOMT activity as described in Materials and Methods. Each value represents the mean ± SEM of determinations from three independent experiments.

View larger version (33K): [in this window] [in a new window]   FIG. 9. Effect of NE on phosphorylated p38MAPK in rat pinealocytes. Pinealocytes (5 x 105 cells/0.5 ml) were cultured for 24 h and treated with NE (10 µM) for different time periods as indicated. The cells were collected by centrifugation, dissolved in 1x sample buffer and analyzed by Western blotting using an antibody against phosphorylated p38MAPK (p-p38MAPK) as described in Materials and Methods. The immunoblot was stripped and reprobed with specific antibodies against total p38MAPK. The blot presented is representative of three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previously we have shown that p38^MAPK inhibitors can inhibit phosphodiesterase activity in rat pinealocytes, resulting in enhanced cyclic nucleotide accumulation (32). The mechanism involved appears to be independent of p38^MAPK inhibition, because SB202474, the compound that is inactive against p38^MAPK, is equally effective in this capacity as SB202190 and SB203580, the two active compounds (32). In contrast, the effect of p38^MAPK inhibitors on AA-NAT appears to be dependent on p38^MAPK inhibition and independent of phosphodiesterase inhibition because the enhancing effect on agonist-stimulated AA-NAT activity is only observed with SB202190 and SB203580, the two active compounds. Moreover, SB202190 remains effective in enhancing AA-NAT activity when cells are maximally stimulated by a membrane-permeable cAMP analog, (Bu)₂cAMP. Taken together, these results suggest that p38^MAPK inhibitors increase agonist-stimulated AA-NAT activity at a post-cAMP step that is involved in the synthesis of AA-NAT.

Signaling pathways activated by members of the MAPK family represent important intracellular mechanisms that transmit extracellular signals into target gene expression (for review, see Refs. 33 and 34). Activated p38^MAPK is known to phosphorylate transcription factors and kinases involved in the regulation of mRNA synthesis (35 ; for review, see Ref. 33). In addition, p38^MAPK has been shown to cause stabilization of mRNAs (33, 36). However, our results indicate that the effect of p38^MAPK inhibition on AA-NAT is probably not due to changes in the transcription of AA-NAT, because neither p38^MAPK inhibitor has an effect on AA-NAT mRNA levels up to 16 h after NE stimulation. In comparison, the two p38^MAPK inhibitors produce a sustained increase in the amount of AA-NAT protein from 8–16 h after NE stimulation. Moreover, p38^MAPK inhibitors increase the maximal fold AA-NAT response that can be elicited by NE or (Bu)₂cAMP. These results point toward a posttranscriptional site of action of p38^MAPK inhibitors on AA-NAT regulation. This is also supported by the observation of an enhancing effect of SB202190 on AA-NAT activity at 6 h, but not at 3 h, after NE or (Bu)₂cAMP stimulation. Posttranscriptional regulation of gene expression by p38^MAPK has previously been demonstrated in other cell types (for review, see Ref. 33).

Another possible mechanism that can account for our observations of the effect of p38^MAPK inhibitors on AA-NAT is that p38^MAPK may regulate the degradation of AA-NAT protein. Degradation of AA-NAT protein has been shown to be controlled by proteasomal proteolysis (3). The rapid decline of AA-NAT that follows removal of NE is blocked by treatment with a proteasomal inhibitor (3). Moreover, complex formation of 14-3-3 with AA-NAT has also been shown to prevent degradation of the AA-NAT enzyme (4). Therefore, it is possible that p38^MAPK inhibitors may reduce the degradation of the AA-NAT protein either by enhancing its binding with 14-3-3 or by reducing proteosomal-dependent AA-NAT degradation. In this regard, activation of other members of the MAPK family has been shown to promote phosphorylation and proteasome-dependent proteolysis (37). Therefore, it would be of interest to determine whether AA-NAT or peptides containing one of the other cyclic nucleotide kinase sites involved in binding to 14-3-3 are phosphorylated by p38^MAPK.

Our results also suggest that during NE stimulation, activation of p38^MAPK, apart from initiation of the transcription and translation of AA-NAT, also sets into motion a series of cellular events that limit the maximal response attainable poststimulation. This is not a new concept, as the inhibitory transcription factor inducible cAMP early repressor has also been shown to be stimulated by NE (38). The degree of enhancement of AA-NAT transcription by CREB phosphorylation (24, 39) is inversely linked to inducible cAMP early repressor, which competes with CREB for CRE sites on the AA-NAT promoter (38, 40). Our findings of p38^MAPK inhibition on AA-NAT add to the importance of this concept by indicating that a negative signal also operates at the translational level and limits the magnitude of the AA-NAT response poststimulation.

Similar to previous studies (41, 42), our results showed that NE treatment causes parallel increases in AA-NAT activity and MT accumulation. However, the increase in NE-stimulated AA-NAT activity produced by p38^MAPK inhibition alone is not accompanied by a corresponding increase in MT production. Our results suggest that the lack of effect on NE-stimulated MT accumulation by p38^MAPK inhibitors could be secondary to a lack of substrate because of the prolonged incubation time, or alternatively, HIOMT may become limiting. In the presence of 5-methoxytryptamine, which acts as an alternate substrate for conversion to MT by AA-NAT, SB202190 has an enhancing effect on NE-stimulated MT production that parallels its effect on NE-stimulated AA-NAT activity, confirming that SB202190 enhances NE-stimulated AA-NAT activity in intact cells. However, we could not exclude the possibility that HIOMT may become limiting even though p38^MAPK inhibition has no effect on HIOMT activity. Interestingly, HIOMT has been reported to drive the photoperiodic changes in the amplitude of the MT peak in Siberian hamsters (43). However, in rats there is only a small nocturnal increase in HIOMT activity (44).

Recently, p38^MAPK has been shown to regulate the oscillation of AA-NAT in the chick pineal gland (45). Our results indicate that p38^MAPK also plays an important role in regulating rat pineal function. This is based on the observations that NE can activate p38^MAPK and that inhibition of p38^MAPK amplifies the NE-stimulated AA-NAT protein and enzymatic activity. Together, these findings suggest that activation of p38^MAPK is part of the negative signal used by the pineal gland to control the magnitude of the AA-NAT response to adrenergic stimulation. To better understand the mechanism by which p38^MAPK affects AA-NAT activity, it will be of interest to distinguish whether inhibition of p38^MAPK has an effect on the translation efficiency or degradation of the AA-NAT protein.

	Acknowledgments

 
We thank Luke Price and Michelle Longson for excellent technical assistance.

	Footnotes

 
This work was supported by grants from the Canadian Institutes of Health Research.

Abbreviations: AA-NAT, Arylalkylamine-N-acetyltransferase; CREB, cAMP response element-binding protein; HIOMT, hydroxyindole-O-methyltransferase; MT, melatonin; NE, norepinephrine.

Received September 5, 2003.

Accepted for publication November 4, 2003.

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