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Originally published In Press as doi:10.1074/jbc.M609943200 on March 14, 2007

J. Biol. Chem., Vol. 282, Issue 18, 13438-13446, May 4, 2007
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Activation of Histidine Decarboxylase through Post-translational Cleavage by Caspase-9 in a Mouse Mastocytoma P-815*

Kazuyuki Furuta{ddagger}, Kazuhisa Nakayama{ddagger}, Yukihiko Sugimoto{ddagger}, Atsushi Ichikawa§, and Satoshi Tanaka1

From the {ddagger}Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan and the Departments of §Physiological Chemistry and Immunobiology, School of Pharmaceutical Science, Mukogawa Women's University, Nishinomiya, Hyogo 663-8179, Japan

Received for publication, October 23, 2006 , and in revised form, January 16, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
L-Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine synthesis in mammals. Although accumulating evidence has indicated the post-translational processing of HDC, it remains unknown what kinds of proteases are involved. We investigated the processing of HDC in a mouse mastocytoma, P-815, using a lentiviral expression system. HDC was expressed as a 74-kDa precursor form, which is cleaved to yield the 55- and 60-kDa forms upon treatment with butyrate. Alanine-scanning mutations revealed that two tandem aspartate residues (Asp517-Asp518, Asp550-Asp551) are critical for the processing. Treatment with butyrate caused an increase in the enzyme activity of the cells expressing the wild type HDC, but not in the cells expressing the processing-incompetent mutant. An increase in histamine synthesis by butyrate was accompanied by formation of the 55- and 60-kDa form of HDC. In addition, the in vitro translated 74-kDa form of HDC was found to undergo a limited cleavage by purified human caspase-9, whereas the alanine-substituted mutants were not. Processing and enzymatic activation of HDC in P-815 cells was enhanced in the presence of a Zn2+ chelator, TPEN. Although treatment with butyrate and TPEN drastically augmented the protease activity of caspase-3, and -9, no apoptotic cell death was observed. Both enzymatic activation and processing of HDC were completely suppressed by a pan-caspase inhibitor, partially but significantly by a specific inhibitor for caspase-9, but not by a caspase-3 inhibitor. These results suggest that, in P-815 cells, histamine synthesis is augmented through the post-translational cleavage of HDC, which is mediated by caspase-9.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Histamine plays diverse roles in physiological and pathological responses, such as inflammation, gastric acid secretion, neurotransmission, and immune modulation, by acting on its specific membrane receptors, H1, H2, H3, and H4 (1–7). L-Histidine decarboxylase (HDC,2 EC 4.1.1.22 [EC] ) is the rate-limiting enzyme for histamine synthesis in mammals, and targeted disruption of mouse HDC gene resulted in the complete loss of de novo synthesis of histamine (8). Several groups purified HDC from various sources including a mouse mastocytoma, P-815, indicating that purified HDC is a homodimer consisting of a 53–55-kDa subunit (9–11). In 1990, Joseph et al. (12) succeeded in cDNA cloning of rat HDC for the first time, and demonstrated that the HDC cDNA encodes a protein, of which the molecular mass is 74 kDa. Subsequent cDNA cloning from P-815 cells revealed that mouse HDC cDNA also encodes a 74-kDa protein (13). These results indicate that HDC is initially translated as a 74-kDa form and then converted into the 53–55-kDa form through the post-translational processing. HDC belongs in the fold type I pyridoxal phosphate-dependent enzyme (13, 14). Although the region around the active lysine residue of HDC shows homology with the other enzymes in this group, such as the aromatic L-amino acid decarboxylase and glutamate decarboxylase, the C-terminal 20-kDa region is unique and contains no homologous sequence with any other protein.

The role of the post-translational processing was first investigated by comparing the recombinant 53- and 74-kDa form of HDC in various expression systems. In the baculovirus-insect cell expression system, the 74-kDa form with a relatively low activity was recovered in the insoluble fraction, while the 53-kDa form in the soluble fraction (15). In vitro digestion of the 74-kDa form in the insoluble fraction of the insect cells by porcine pancreatic elastase resulted in liberation of the soluble 53-kDa form, which exhibited a similar characteristics to the purified enzyme (16). We also found that the 74-kDa form of HDC is accumulated in the ER in COS-7 cells while the 54-kDa mutant lacking the C-terminal region is in the cytosol (17). Although the 74-kDa form of HDC has neither the N-terminal signal sequence nor hydrophobic membrane anchor, the in vitro translated 74-kDa HDC protein was post-translationally targeted to the microsomal fraction, which was mediated by the C-terminal 20-kDa region (17). These results indicate that the C-terminal 20-kDa region mediates the ER-targeting of HDC and the membrane-bound 74-kDa form possesses a lower activity than that of the truncated 54-kDa form in the cytosol. Fleming and Wang (18) demonstrated that rat 74-kDa form expressed in COS-7 cells is cleaved to multiple forms and that formation of the main 55-kDa form is prevented by mutations at the residues, Ser502, Lys503, and Asp504, whereas these mutations had no effects on the enzyme activity. However, this putative processing site was not found in the primary sequence of mouse and human HDC, and no processed forms were detected in COS-7 cells expressing mouse 74-kDa HDC (17). The processing site of mouse HDC remains to be clarified, especially in histamine-forming cells.

Preparation of specific antibodies against HDC has shed light on the processing patterns of endogenous HDC in various sources. We previously demonstrated by immunoprecipitation of the nascent 35S-labeled HDC that the 74-kDa form is localized in the cytosol and ER while the 53-kDa form in the granule fraction in a rat mast cell line, RBL-2H3 (19). In this cell line, histamine synthesis was detected both in the cytosol and in the granule fraction. We have recently found that activated mouse neutrophils have a potential to produce histamine (20). Post-translational processing of HDC detected by immunoprecipitation of elicited neutrophils was found to be rapid, and only 53-kDa form was detected by immunoblot analysis. The 53-kDa form was co-localized with matrix metalloproteinase-9, which is a granule enzyme of neutrophils. On the other hand, only 74-kDa form was detected in lipopolysaccharide-activated mouse macrophages and monomeric IgE-activated immature bone marrow-derived cultured mast cells, in both of which a large increase in histamine synthesis is observed (21, 22). Other variants with intermediate sizes (58~64-kDa) have also been reported in the stomach, fetal liver, and testis (18, 23, 24). These findings have highlighted the complexity in the regulation of enzyme activity and intracellular localization of HDC through the post-translational processing.

The purpose of this study is to identify the processing site and the responsible protease(s) for the post-translational processing of mouse HDC in histamine-forming cells. We also investigated whether enzymatical activation of HDC occurs upon its post-translational processing. We performed a series of experiments to address these problems using the lentiviral expression system with the mouse mastocytoma, P-815, because the 53-kDa form of HDC was initially purified from this cell line, indicative of the expression of the processing enzyme.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The following materials were purchased from the sources indicated: an anti-FLAG monoclonal antibody (clone M2), N,N,N', N'-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN), sodium butyrate, polybrene, and recombinant mouse granzyme B from Sigma, an anti-HA monoclonal antibody (clone 3F10) from Roche Diagnostics (Mannheim, Germany), an anti-caspase-9 antibody from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), an anti-actin antibody form Chemicon (Temecula, CA), Ac-DEVD-AMC and Ac-LEHD-AMC from Peptide Institute Inc. (Osaka, Japan), benzyl-oxycarbonyl-Val-Ala-D L-Asp-fluoromethylketone (z-VAD-fmk), z-DEVD-fmk, z-LEHD-fmk, and recombinant human caspases (caspase-1, -2, -3, -6, -7, -8, -9, and -10) from Calbiochem, L-[35S]methionine from PerkinElmer (Wellesley, MA), T7 RNA polymerase, and the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA), Flexi Rabbit Reticulocyte Lysate system from Promega (Madison, WI), Lipofectamine 2000 reagent, ViraPower Lentiviral Expression System, and FITC annexin V from Invitrogen (Carlsbad, CA), horseradish peroxidase-conjugated secondary antibodies against rabbit IgG, rat IgG, and mouse IgG from Dako (Glostrup, Denmark), an ECL Western blot detection reagent from Amersham Biosciences. All other chemicals were commercial products of reagent grade.

Construction of Expression Vectors—Plasmid pBSII/3FLAG-HDC-HA was constructed by inserting the oligonucleotide pairs coding 3x tandem repeats of the FLAG epitope following the initiating methionine (MDYKDHDGDYKDHDIDYKDDDDK) at the 5' terminus of mouse HDC cDNA and the oligonucleotide pairs coding the HA epitope (YPYDVPDYA) at the 3' terminus of mouse HDC. To prepare the pLenti/3FLAG-HDC-HA, the pBSII/3FLAG-HDC-HA was digested and subcloned into the pLenti empty vector.

Site-directed Mutagenesis—Plasmid pBSII/3FLAG-HDC-HA was used as a template to perform mutagenesis using QuikChange site-directed mutagenesis kit according to the manufacturer's instruction. Synthetic oligonucleotides containing alanine substitution were used to introduce the site-directed mutations. Mutations were confirmed by DNA sequencing. To prepare the lentivirus expression vectors, the insert of pLenti/3FLAG-HDC-HA was replaced with the mutated insert as described above. Details of constructions used in the alanine-scanning mutagenesis are summarized in Table 1.


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  		TABLE 1
Alanine substitution of HDC

Alanine-scanning mutagenesis was performed to identify the cleavage sites of HDC. The amino acid residue mutated to alanine in each construct is presented.

 
Cell Culture and Butyrate Treatment—293FT cells were grown in Dulbecco's modified Eagle's medium containing 4.5 mg/ml glucose, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 0.5 mg/ml G418, and 10% heat-inactivated fetal calf serum at 37 °C in a fully humidified 5% CO2 atmosphere. A mouse mastocytoma cell line, P-815, was grown in RPMI 1640 medium containing 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated fetal calf serum (complete RPMI medium) at 37 °C in a fully humidified 5% CO2 atmosphere. P-815 cells (2.0 x 105 cells/ml) were incubated for 24 h at 37 °C in the complete RPMI medium with or without butyrate and TPEN in the indicated concentrations.

Lentiviral Expression—The lentiviral vector, pLenti/3FLAG-HDC-HA, and its mutants were introduced using a ViraPower Lentiviral Expression system according to the manufacturer's instruction. Briefly, 293FT cells were co-transfected with pLenti/3FLAG-HDC-HA and ViraPower Packaging Mix using Lipofectamine 2000 reagent to generate the recombinant lentivirus. The medium was replaced 24 h after the initial transfection, and was collected at Day 3 as the medium containing the recombinant lentivirus. P-815 cells were incubated in the presence of the viral solutions containing 6 µg/ml of polybrene for 24 h at 37 °C. The infected cells were selected by 2.5 µg/ml blasticidin S.

Immunoblot Analysis—Immunoblot analyses were performed as described previously (24). Antibodies raised against the FLAG epitope, the HA epitope, caspase-9, and actin were used as the primary antibodies, and horseradish peroxidase-conjugated antibodies raised against mouse IgG, rat IgG, and rabbit IgG were used as the secondary antibodies. The membranes were stained using an ECL kit according to the manufacturer's instruction.

Histidine Decarboxylase Assay—P-815 cells were washed in phosphate-buffered saline and were lysed in 50 mM HEPES-NaOH pH 7.3 containing 0.2 mM dithiothreitol, 0.01 mM pyridoxal 5'-phosphate, 2% polyethylene glycol 300, 0.2 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. The lysate was centrifuged at 20,000 x g for 30 min at 4 °C, and the resultant supernatant was subjected to the enzyme assay for HDC. The assay was performed in 1 ml of reaction mixture (100 mM potassium phosphate pH 6.8, 0.2 mM dithiothreitol, 0.01 mM pyridoxal 5'-phosphate, and 2% polyethylene glycol 300) in the presence of 0.8 mML-histidine for 4 h at 37°C. The histamine formed was extracted and separated on a cation exchange column, WCX-1 (Shimadzu, Kyoto, Japan), by HPLC and then fluorometrically measured by the o-phthalaldehyde method (25).

In Vitro Translation and Cleavage Assay—Radiolabeled HDC was prepared by in vitro transcription and translation using [35S]methionine. Plasmid pBSHDC was linearized with SalI, and RNA was transcribed using T7 RNA polymerase as described (17). In vitro translation was performed using Flexi Rabbit Reticulocyte Lysate System. Translation reaction mixture, using 1.0 µg of RNA in 100 µl reaction volume, was incubated for 30 min at 30 °C. The labeled translation product (7.5 µl) was incubated with 15 µl of recombinant human caspases (15 units), granzyme B (90 units) or the P-815 cell lysate (10 mg protein/ml) in 25 mM HEPES-KOH pH 7.4 containing 100 mM KCl, 5 mM dithiothreitol, and 0.1% CHAPS for 3 h at 37 °C. The reaction was terminated by addition of SDS-sample buffer. Each sample was boiled for 3 min and was subjected to SDS-PAGE. The radiolabeled HDC protein was detected by autoradiography.

Measurement of Caspase Activity—Caspase-3- and caspase-9-like activities were measured using the fluorogenic substrates, Ac-DEVD-AMC and Ac-LEHD-AMC, respectively. P-815 cells were washed twice with phosphate-buffered saline and were lysed in 50 mM HEPES-KOH pH 7.4 containing 150 mM NaCl, 0.5% Triton X-100 and 0.2 mM phenylmethylsulfonyl fluoride. The lysate was centrifuged at 20,000 x g for 5 min at 4 °C. Protein concentrations of the supernatants were measured and adjusted at 0.2 mg of protein/ml. The assay was performed in 96-well plate by incubating 20 µg of cell lysate in 200 µl of reaction buffer (100 mM HEPES-KOH pH 7.4, 10% glycerol, and 5 mM dithiothreitol) in the presence of 50 µM of each fluorogenic substrate. Release of 7-amino-4-methylcoumarin (AMC) was measured after 4 h of incubation at 37 °C by microplate reader (Wallac ARVO sx-1420, PerkinElmer) with excitation and emission wavelengths of 380 nm and 460 nm, respectively.

Flow Cytometry—Apoptotic cell death was evaluated by measuring the translocation of phosphatidylserine in the plasma membrane using the FACSCalibur (Becton Dickinson) with a FITC-conjugated annexin and propidium iodide. Apoptotic cell death of P-815 cells were confirmed when the cells were incubated for 12 h in the presence of 1 µM staurosporine.


Figure 1
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  		FIGURE 1.
Post-translational processing of HDC upon butyrate treatment. Wild type P-815 cells (None) or the cells expressing the epitope-tagged HDC (HDC) were incubated at 37 °C for 24 h in the presence (+) or absence (–) of 3 mM butyrate. The cell lysate was subjected to immunoblot analyses using an antibody against the N-terminal FLAG epitope (A, 1:3000) or the C-terminal HA epitope (B, 1:500). Arrows indicate the position of the 74-kDa form of HDC, while closed arrowheads indicate those of the processed forms. Open arrowheads indicate the bands that appear in a non-reproducible fashion. Asterisks indicate the nonspecific signals, which were also detected in the parental P-815 cells.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Post-translational Cleavage of HDC in P-815 Cells Treated with Butyrate—We previously purified HDC as a homodimer consisting of a 53-kDa subunit from P-815 cells, which were maintained in peritoneal cavity of the BDF1 mouse (11). This cavity incubation was found to induce the expression of HDC in P-815 cells at the transcriptional level (26). Because a prolonged culture of P-815 cells resulted in a decrease in the HDC level, we investigated post-translational processing of HDC expressed using a lentiviral expression in these cells. This approach allowed us to evaluate the processing of only exogenously expressed HDC without the contribution of endogenous HDC and histamine synthesis (HDC activity <0.01 pmol/min/mg protein).

Lentivirally transduced epitope-tagged HDC was expressed in P-815 cells as a 74-kDa form, which was detected by both antibodies against the N-terminal (FLAG) and the C-terminal (HA) tags (Fig. 1). Because several effects of butyrate on mast cell function have been found, such as histamine and heparin biosynthesis (27–29), we investigated the effects of butyrate on the molecular mass of HDC. In addition to the 74-kDa form, three forms with smaller molecular masses were detected in the cells treated with butyrate using the N-terminal anti-FLAG antibody (Fig. 1). We could detect no smaller fragments using the C-terminal anti-HA antibody. These results suggest that the post-translational processing of HDC yields 46-, 55-, and 60-kDa forms by accompanying a rapid degradation of the C-terminal fragment. Because a series of C-terminal deletion mutants of HDC, HDC1–476, HDC1–460, HDC1–420, and HDC1–380, exhibited no enzyme activity (data not shown), it seems likely that 46-kDa form of HDC has no enzymatic activity. We, therefore, focused on the generation of two major processed forms, 55- and 60-kDa forms. Because transduced HDC contains the epitope tags of about 2 kDa, the 55- and 60-kDa forms should correspond to 53- and 58-kDa forms of the endogenous HDC.

Determination of the Cleavage Site by Alanine-scanning Mutagenesis—We performed a study using a series of alanine-substituted mutants to identify the amino acid residues critical for the cleavage of HDC in P-815 cells (Table 1). Generation of the 60-kDa form of HDC was abolished in two triple alanine mutants, DPF547–549 and DDC550–552, and in a double mutant, DD550–551, whereas not in a triple mutant TMP544–546 (Fig. 2A). Mutations of individual amino acids revealed that an alanine substitution of Pro548 or Asp550 partially suppressed the generation of the 60-kDa form upon treatment with butyrate (Fig. 2B). On the other hand, generation of 55-kDa form was completely abolished by a single mutation at the residue, Asp518 or Pro519 (Fig. 2C). A single alanine mutation at the residue Asp517 was also found to suppress the formation of 55-kDa form.

Activation of HDC through the Post-translational Processing—Because butyrate was reported to augment histamine synthesis in P-815 cells, we investigated the relationship between the processing and enzyme activity of HDC. The butyrate-induced increase in histamine synthesis was found to be accompanied by formation of 55- and 60-kDa form of HDC (Fig. 3, A and B). The amount of the 74-kDa form was not changed during the treatment, whereas that of the processed forms was increased. Densitometric measurement exhibited a good correlation between the amount of the processed forms and the enzyme activity. Furthermore, the C-terminal deletion mutants, which correspond to the 55- and 60-kDa form of HDC, exhibited higher specific activity when compared with the 74-kDa form (~8-fold, Fig. 3, C and D). We then constructed an HDC mutant that is resistant to cleavage in P-815 cells treated with butyrate to investigate the effects of the post-translational processing on the enzyme activity. The mutant form, in which aspartate residues at 518, 550, and 551 are substituted to alanine, was found to be completely resistant to the butyrate-induced cleavage (Fig. 3E). The butyrate treatment increased the enzyme activity in P-815 cells expressing the wild type HDC, but not in the cells expressing this mutant (Fig. 3F). These results indicate that enzymatic activation of HDC upon the butyrate treatment requires the post-translational processing. Activation of endogenous HDC was also measurable upon the butyrate treatment in the parental cells, indicating that the increase in the enzyme activity in the cells expressing the recombinant HDC was partially because of the activation of endogenous HDC. However, its contribution was negligible, because the levels of enzyme activation were very small in the parental cells (0.100 ± 0.0211 pmol/min/mg protein, in the presence of 3 mM butyrate, n = 3).


Figure 2
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  		FIGURE 2.
Determination of the cleavage site of HDC by alanine-scanning mutagenesis. P-815 cells were infected with the recombinant lentiviruses encoding the alanine-substituted HDC mutants described in Table 1, respectively. Selected cells were incubated with 3 mM butyrate at 37 °C for 24 h. The cell lysate was subjected to immunoblot analyses using an antibody against the FLAG epitope. Arrows indicate the position of the 74-kDa form of HDC, while arrowheads indicate those of the processed 55-kDa and 60-kDa forms. Alanine substitution was performed at the amino acid residues, cleavage at which results in formation of 55-kDa (C) or 60-kDa form (A and B).

 
Cleavage of in Vitro Translated HDC by Purified Proteases—Investigation of cleavage sites of HDC by alanine-scanning mutagenesis raised a possibility that HDC is post-translationally cleaved at the aspartic residues. Because caspase family and granzyme B have been found to require an aspartate residue at the cleavage site, we then investigated whether purified caspase or granzyme B has a potential to cleave HDC protein in vitro.A band, of which molecular mass is ~53-kDa, was detected when the 35S-radiolabeled 74-kDa form of HDC was incubated with caspase-9 (Fig. 4). Thus, a single processed form was generated in vitro, whereas two different processed forms were detected in P-815 cells expressing epitope-tagged HDC upon treatment with butyrate. This discrepancy may be attributed to differences of the substrate/protease ratio between in vitro and in cells. Although purified mouse granzyme B also generated three processed species, no processed forms were found in the 50–60-kDa ranges. We then performed the in vitro cleavage analyses with the alanine-mutated HDC to identify the amino acid residues recognized by purified caspase-9. The processing-resistant mutant, HDCD518/550/551, was partially resistant to cleavage by caspase-9 (Fig. 5A). An additional mutation at the aspartate residue 517 of HDC was found to completely abolish this limited cleavage. Because human caspase-9 is highly homologous to mouse caspase-9 and purified human caspase-9 can compensate for the loss of mouse caspase-9 in the cell extracts of the caspase-9-deficient mice (30), it is conceivable that mouse caspase-9 can also cleave HDC in a similar fashion. As a negative control, no cleavage was found in the presence of caspase-3 (Fig. 5B).


Figure 3
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  		FIGURE 3.
Activation of HDC through post-translational processing. A and B, P-815 cells expressing the epitope-tagged HDC were incubated with 3 mM butyrate at 37 °C for the indicated periods. The cell lysate was subjected to immunoblot analyses using an antibody against the FLAG epitope and actin, respectively. The relative intensity of each immunoreactive band was densitometrically determined and presented. The enzymatic activity for HDC of the same sample was measured. C and D, P-815 cells expressing the epitope-tagged wild-type HDC (WT) or its C-terminal deletion mutants (HDC1–551 and HDC1–518) were incubated at 37 °C for 24 h in the presence (+) or absence (–) of 3 mM butyrate. Immunoblot analysis and enzyme assay were performed as described above. (E and F) P-815 cells expressing the epitope-tagged wild type HDC (WT) or the alanine-substituted mutant (HDCD518/550/551) were incubated at 37 °C for 24 h in the presence (+) or absence (–) of 3 mM butyrate. Immunoblot analysis and enzyme assay were performed as described above. An arrow indicates the position of the 74-kDa form of HDC, while arrowheads indicate those of the 55- and 60-kDa forms (A, C, and E). The values were represented as the means ± S.E. (n = 3, B, D, and F).

 
Effects of a Zn2+ Chelator, TPEN, on Activation of HDC—Caspase-3 was shown to be enzymatically activated by butyrate in a human mast cell line, HMC-1, and this activation was enhanced in the presence of a Zn2+ chelator, TPEN (31). Because Zn2+ has a potential to inhibit cysteine proteases including caspases, we then investigated the effects of TPEN on activation of the caspase cascade and HDC in P-815 cells. Treatment of the cells with TPEN was found to significantly enhance the specific cleavage and enzymatic activation of HDC induced by butyrate (Fig. 6, A and B). Contribution of endogenous HDC to the total activity was negligible, because only a slight increase in the activity was observed in the parental cells (0.157 ± 0.0195 pmol/min/mg protein, in the presence of 3 mM butyrate and 3 µM TPEN, n = 3). Furthermore, treatment with butyrate in combination with TPEN caused a drastic increase in the enzyme activity of caspase-9, as well as that of caspase-3 (Table 2). Expression of caspase-9 in P-815 cells was confirmed by immunoblot analysis using its specific antibody (Fig. 6C). Proteolytic conversion of caspase-9 was observed in the cells treated with butyrate in combination with TPEN, which is in agreement with enzymatic activation of caspase-9.


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  		TABLE 2
Effects of butyrate and TPEN on enzyme activity of caspase-3 and -9

P-815 cells were incubated at 37°C for 24 h with or without 3 mM butyrate in the presence or absence of 3 µM TPEN. Caspase activities were measured using the specific fluorogenic substrates as described under "Experimental Procedures." Each caspase activity is presented by the fold increase (enzyme activity in control = 1.00). The results are represented as the means ± S.E. (n = 3).

 
The drastic activation of the caspase cascade in P-815 cells treated with butyrate raised a possibility that treatment with butyrate induces apoptotic cell death, especially in the presence of TPEN. We evaluated the apoptotic cell death by detecting translocation of phosphatidylserine in the plasma membrane with fluorescent annexin V. No increase in apoptotic cell death was observed in the cells treated with butyrate even in the presence of TPEN (Fig. 6D). This result indicates that activation of the caspase cascade induced by butyrate does not lead to the apoptotic cell death of P-815. Prolonged treatment (48 and 72 h) with butyrate did not result in increase in the apoptotic cell death (data not shown).


Figure 4
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  		FIGURE 4.
Cleavage of in vitro translated HDC by purified proteases. Mouse HDC protein labeled with [35S]methionine was prepared by in vitro transcription and translation. The aliquots of radiolabeled HDC were incubated at 37 °C for 3 h in the presence and absence (None) of human-purified caspase (hCaspase)-1, -2, -3, -6, -7, -8, -9, -10, or mouse-purified granzyme B (Gzmb). The cleavage pattern was analyzed by SDS-PAGE and autoradiography. The radiolabeled substrate was loaded without incubation as a control (Control). An arrow indicates the 74-Da form, while an arrowhead indicates the 53-kDa form. The asterisks indicate the other processed forms.

 


Figure 5
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  		FIGURE 5.
Effects of alanine substitution on in vitro cleavage of HDC by caspase-9. In vitro cleavage assay was performed as described in the legend to Fig. 4. [35S]methionine labeled wild type HDC protein and its mutants (HDCD518/550/551 and HDCD517/518/550/551) were incubated with the indicated concentrations of human purified caspase-3 (B), or -9 (A). An arrow indicates the 74-Da form, while an arrowhead indicates the 55-kDa form.

 
Suppression of Post-translational Processing of HDC by Specific Caspase Inhibitors—We then investigated the effects of specific inhibitors for caspases on the cleavage and activation of HDC. Pretreatment of the cells with a pan-caspase inhibitor, z-VAD-fmk, was found to completely abolish the processing of HDC upon butyrate treatment. Pretreatment with a caspase-9 inhibitor, z-LEHD-fmk, partially suppressed the conversion of HDC (Fig. 7A). In agreement with these results, enzymatic activation of HDC was abolished by the pan-caspase inhibitor, and partially but significantly suppressed by the caspase-9 inhibitor (Fig. 7B). These inhibitors themselves exhibited no inhibitory effects on the catalytic activity of HDC (data not shown). A specific inhibitor for caspase-3, z-DEVD-fmk, was found to have no effects on the cleavage and activation of HDC.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We investigated in this study how the post-translational processing of HDC is regulated in histamine-forming cells. Although we previously showed that in vitro cleavage by elastase of the 74-kDa form, which exhibited a low enzyme activity, resulted in generation of an active 53-kDa form (16), it remains to be clarified whether enzymatical activation occurs upon post-translational processing of HDC in histamine-forming cells. In expression systems, mouse and rat 74-kDa form of HDC were found to exhibit no or little enzyme activity, although enzyme activity of human 74-kDa form was reported to be equivalent to that of the 54-kDa C-terminal deletion mutant (15, 18, 32). Our results strongly indicate that the proteolytic conversion itself plays a critical role in enzymatic activation of HDC, at least in the mouse mastocytoma cell line. On the other hand, recent studies suggested that the 74-kDa form is responsible for histamine synthesis in activated macrophages and immature mast cells (21, 22). A simple hypothesis that enzymatic activation of HDC occurs through its post-translational processing may not be applicable to all the sources that produce histamine. Further studies are required to address how histamine synthesis is effectively catalyzed by the 74-kDa form in some kinds of cells.

The alanine-scanning mutagenesis revealed that the two regions containing diaspartate residues are critical for the cleavage of HDC, and the subsequent analyses identified caspase-9 as the potential candidates for the processing enzyme. In vitro cleavage analysis indicates that caspase-9 can directly cleave the HDC protein, not through activation of caspase-3. Regarding the cleavage of HDC, the involvement of ubiquitin-proteasome system and m-calpain have been reported in a rat mast cell line and in vitro, respectively (33, 34), although these cleavages lead to degradation and inactivation of HDC. Our results strongly suggest that mouse HDC is enzymatically activated upon the limited cleavage by caspase-9 in a mastocytoma, P-815. Fleming et al. (35) recently demonstrated that the amino acid sequence 617–633 in rat HDC, which is highly conserved between rat and mouse, plays an inhibitory role in catalytic reaction, which also supports the concept of the enzyme activation by its C-terminal truncation.

Accumulating evidence has indicated that caspases recognize at least four contiguous amino acids in their substrates, P4-P3-P2-P1, and an aspartate residue is absolutely required in the P1 position (36). Thornberry et al. (37) previously determined the substrate specificity of the caspase family and granzyme B by a combinatorial approach, depicting several characteristics of the preferred substrates. They showed the strict requirement of glutamate in the P3 position in case of caspase-9. Putative motifs for processing of HDC identified in the current study, EGGDD (514–518) and DPFDD (547–551), are not compatible with the combinatorially determined motifs for caspase-9. However, few proteins have been identified as the substrates for caspase-9, and several exceptions to this prediction have also been reported; the cleavage motifs of caspase-9 in vimentin (IDVD), are dissimilar to the combinatorially determined motifs (38). The cleavage site should be the tandem aspartate residues, 517/518, when caspase-9 works as the processing enzyme for HDC. Because we could not detect a larger processed form under our in vitro cleavage assay condition, it remains to be clarified whether caspase-9 is involved in the formation of the 60-kDa form. The two putative motifs for the processing are highly conserved among mouse, rat and human, raising the possibility that caspase-9 is also involved in the processing of HDC in rat and human (12, 13, 39).


Figure 6
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  		FIGURE 6.
Effects of a Zn2+ chelator, TPEN, on activation of HDC. P-815 cells expressing the epitope-tagged HDC were incubated at 37 °C for 24 h in the presence or absence of 3 mM butyrate and 3 µM TPEN as indicated. A, cell lysate was subjected to immunoblot analyses using an antibody against the FLAG epitope. An arrow indicates the 74-kDa form of HDC, while arrowheads indicate the 55- and 60-kDa forms. The relative intensity of each immunoreactive band was densitometrically determined and presented. B, enzymatic activity of the same sample was measured. The values were represented as the means ± S.E. (n = 3). *, p < 0.01 is regarded as significant by the Student's t test (versus (-)butyrate, (–)TPEN). C, immunoblot analysis was performed using an antibody raised against caspase-9 (1:200). An arrow indicates the pro-form of caspase-9, while an arrowhead indicates the processed form of caspase-9. The amount of actin was measured as a loading control (anti-actin, 1:1000). D, apoptotic cell death was detected by monitoring the translocation of phosphatidylserine in the plasma membrane, which is detected by FITC-conjugated annexin V. Flowcytometric analysis was performed using the FACSCalibur. The population of each area (% of total event number) is presented in the right panel. Positive control of apoptotic cell death is prepared by incubating P-815 cells with 1 µM staurosporine for 12 h at 37 °C.

 
The caspase family can be classified into two categories, inflammatory and apoptotic caspases, and the latter group can be further divided into initiator and effector caspases (36). Caspase-9 is categorized into the initiator caspases that can activate the downstream effector caspases, such as caspase-3, through the limited cleavage. Although caspase-3 and -9 were drastically activated upon treatment with butyrate, especially in the presence of TPEN, no increase in apoptotic cell death was observed in P-815 cells. Accumulating evidence suggests that activation of the caspase cascade do not always result in apoptotic cell death. Recent studies have revealed diverse roles of the initiator caspases independently of apoptosis; caspase-9 was implicated in erythroid differentiation and proplatelet formation (40, 41). Because the substrates for caspase-9 have not been identified in these reports, it remains to be clarified how caspase-9 regulate these cellular responses. Suppression of cleavage and enzymatical activation of HDC by the specific peptide inhibitors for caspases indicates that casapase-9 is required for full activation of HDC. Many known caspase substrates are just cleaved as bystanders, because they happen to contain a caspase cleavage site in their sequence. Only a few gain-of-function cases have been reported about the cleavage by caspases (38). In contrast, we can conclude that the processing of HDC by caspase-9 clearly contribute to the increased de novo synthesis of histamine that usually occurs during mast cell maturation.

Butyrate has been reported to function as an inhibitor for histone deacetylase, thereby inducing histone hyperacetylation and concomitant transcriptional activation of several sets of genes (42, 43). Histone deacetylase inhibitor has been reported to cause growth arrest and differentiation of various kinds of cells. Previous studies indicated that butyrate induces growth arrest, increase in histamine and heparin biosynthesis, and granule maturation in cultured mast cell lines including P-815 (27–29, 44). These reports are consistent with our results showing that histamine synthesis is augmented upon butyrate treatment. Butyrate is also known to induce apoptotic cell death in several kinds of cells, such as colon carcinoma, T-cell lymphoma, and erythroleukemia cells (45–47). Recently, Ho et al. (31) reported that apoptotic cell death was observed in a human mast cell line, HMC-1, treated with the combination of butyrate and TPEN, with a drastic enzymatic activation of caspase-3. We detected the significant increase in enzyme activity of caspase-3 and -9 in P-815 cells treated with butyrate alone, and this induction was further augmented in the presence of TPEN. However, no apoptotic cell death was observed in our system with P-815 cells. The increase in the enzyme activity of caspases may not fulfill the criterion required for induction of apoptosis in P-815 cells, because activation of caspases was found to be transient and transcription of genes of caspase-3 and -9, was all suppressed upon butyrate treatment (data not shown). This transient and limited activation of the caspase cascade may contribute to differentiation of P-815 cells.


Figure 7
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  		FIGURE 7.
Suppression of the post-translational processing of HDC by specific caspase inhibitors. P-815 cells expressing the epitope-tagged HDC were pretreated with or without (vehicle, Me2SO) 100 µM of various caspase inhibitors (pan-caspase; z-VAD-fmk, caspase-3; z-DEVD-fmk, caspase-9; z-LEHD-fmk) for 24 h at 37 °C. The cells were then stimulated with 3 mM butyrate for 24 h at 37 °C. A, cell lysate was subjected to immunoblot analyses using an antibody against the FLAG epitope and actin. An arrow indicates the position of the 74-kDa form of HDC, while arrowheads indicate the 55- and 60-kDa forms. B, enzymatic activity for HDC was measured. The values were represented as the means ± S.E. (n = 3). *, p < 0.01 (versus vehicle) is regarded as significant by the Student's t test.

 
In summary, caspase-9 was found to play a critical role in activation of HDC in a mouse mastocytoma. Recent studies have shed light on novel roles of histamine in immune system, such as regulation of autoimmune responses, and migration of several leukocytes (7, 48–49). We propose here that caspase-9 is one of novel targets for development of therapeutic approaches for histamine-related diseases.


   FOOTNOTES
 
* This study was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan, and the Takeda Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Immunobiology, School of Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Hyogo 663-8179, Japan. Tel.: 81-798-45-9958; E-mail: s_tanaka{at}mukogawa-u.ac.jp.

2 The abbreviations used are: HDC, L-histidine decarboxylase; AMC, 7-amino-4-methylcoumarine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio] propanesulfonic acid; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter; PI, propidium iodide; TPEN, N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine; z, benzyloxycarbonyl; fmk, fluoromethylketone; HA, hemagglutinin. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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