2014 年 37 巻 1 号 p. 81-86
Mast cells are involved in various immunological responses, although it remains unknown how their terminal differentiation is regulated. We previously established a culture model that mimics the process of mast cell maturation in the cutaneous tissue and found that growth factor independent 1 (Gfi1) was up-regulated whereas its paralogue Gfi1b down-regulated. Here we investigated the roles of Gfi1 and Gfi1b in the process of mast cell maturation using a murine mast cell line, MC9. Gfi1 and Gfi1b cDNAs were stably expressed in MC9 cells using the recombinant lentivirus. Histamine synthesis was significantly induced by stem cell factor (SCF) alone, whereas tryptase expression was significantly augmented in the presence of both SCF and Swiss 3T3 cells. Since exogenously expressed Gfi1 and Gfi1b might affect their expression levels in MC9 cells, we investigated the relationship between the expression profiles of Gfi1/Gfi1b proteins and maturation indices, such as histamine synthesis and tryptase expression. The comparison suggested that histamine synthesis during the co-culture period was positively regulated by Gfi1b while augmented expression of tryptase was abolished by one-sided expression of Gfi1/Gfi1b. Our findings indicated the involvement of Gfi1 and Gfi1b in the process of murine mast cell maturation.
Mast cells are originated in hematopoietic stem cells in the bone marrow and distributed in nearly all vascularized tissues. Accumulating evidence suggests that mast cells are involved in a wide variety of immunological responses in addition to immediate allergy and protection from parasite infection.1) When we try to identify the physiological roles of mast cells, it is critical to characterize the nature of local mast cells, because mast cells undergo terminal differentiation in the tissues, in which they are ultimately resident.2) Interleukin-3 (IL-3)-dependent bone marrow-derived cultured mast cells (BMMCs) are one of the most popular murine culture models, although there are many differences between BMMCs and tissue mature mast cells. Levi-Schaffer et al. established a model more similar to mature mast cells distributed in the connective tissues, in which BMMCs were co-cultured with Swiss 3T3 fibroblasts.3) We modified this method to establish a novel culture model, which shares many characteristics with cutaneous mast cells.4) In our model, BMMCs were co-cultured with Swiss 3T3 cells in the presence of stem cell factor (SCF) for 16 d. During this process, cultured mast cells acquired many characteristics of mature cutaneous mast cells, such as potentials of degranulation in response to substance P and compound 48/80, and enhanced expression of granule proteases including chymase, tryptase, and carboxypeptidase A. Since these changes observed during the co-culture period reflected at least in part the process of mast cell maturation, we then investigated the gene expression profiles by microarray analysis. We focused on growth factor independent 1 (Gfi1) and its paralogue Gfi1b in this study, because Gfi1 was found in the up-regulated genes in the early phase whereas Gfi1b in the down-regulated genes during the co-culture period, indicating the involvement of these transcription factors in mast cell maturation.
Gfi1 and Gfi1b are transcription repressors, which play important roles in growth and differentiation of various hematopoietic lineages.5) Gfi1 was found to play critical roles in development and function of hematopoietic stem cells,6,7) B cells,8) T cells,9) neutrophils,10–12) dendritic cells,13) and macrophages.12) On the other hand, Gfi1b was found to be essential for erythrocyte development.14) However, it remains unknown whether Gfi1 and Gfi1b are involved in regulation of mast cell differentiation and function. In this study we investigate the roles of Gfi1 and Gfi1b using a murine interleukin (IL)-3-dependent mast cell line, MC9, which was co-cultured with Swiss 3T3 in the presence of SCF.
The following materials were purchased from the sources indicated; an anti-Gfi1 antibody, anti-Gfi1b antibody and anti-Lyn antibody from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.), anti-actin antibody from Millipore (Bedford, MA, U.S.A.), recombinant mouse IL-3 from R&D systems (Minneapolis, MN, U.S.A.), tryptase substrate S-2288 from Chromogenix (Mölndal, Sweden), ViraPower Lentiviral Expression System and blasticidin S from Invitrogen (Carlsbad, CA, U.S.A.), mitomycin C from Sigma (St. Louis, MO, U.S.A.), horseradish peroxidase-conjugated secondary antibodies against rabbit immunoglobulin G (IgG) and mouse IgG from Dako (Carpinteria, CA, U.S.A.). All other chemicals were commercial products of reagent grade. Recombinant mouse SCF was prepared using the baculovirus expression system as described previously.15)
Preparation of Expression Vectors and LentivirusesMouse Gfi1 and Gfi1b cDNAs were subcloned by reverse transcription polymerase chain reaction (RT-PCR) using the specific primer pairs. They were inserted in pLenti6 vector. Recombinant lentiviruses were prepared using ViraPower Lentiviral Expression System as described previously.16)
Cell Culture and Lentiviral ExpressionConnective tissue type cultured mast cells derived from murine bone marrow cells were prepared as described previously.4) An IL-3-dependent murine mast cell line, MC9, was grown in RPMI-1640 containing 10% heat inactivated fetal bovine serum (FBS), 0.1 mM nonessential amino acid, 100 units/mL penicillin, 0.1 mg/mL streptomycin, 50 µM β-mercaptoethanol and 1 ng/mL of IL-3 in fully humidified 5% CO2 atmosphere. For lentiviral transduction, MC9 cells were incubated in the presence of the recombinant virus solutions containing 6 µg of polybrene for 24 h at 37°C. The infected cells were then concentrated by selection with 2.5 µg/mL blasticidin S.
Co-culture of MC9 with Swiss 3T3 CellsSwiss 3T3 fibroblasts were seeded with 50% confluency in the same medium without IL-3, treated with 3 µg/mL mitomycin C for 3 h, and further incubated for 3 h in the fresh medium without mitomycin C. MC9 cells were seeded on this Swiss 3T3 cells and co-cultured for 24 h or 48 h in the presence of SCF (30 ng/mL).
Measurement of Histamine Content and L-Histidine Decarboxylase (HDC) ActivityMC9 cells were lysed in 10 mM potassium phosphate, pH 6.8, containing 10 mM KCl, 1.5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 0.2 mM dithiothreitol, 0.01 mM pyridoxal 5′-phosphate, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM benzamidine, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL, E-64, 1 µg/mL pepstatin A and 0.1% Triton X-100, and incubated for 15 min on ice. The lysate was centrifuged at 10000×g for 15 min and the resultant supernatant was subjected to measurement of histamine content and HDC activity as described previously.16)
Immunoblot AnalysisMC9 cells were lysed in 20 mM Tris–HCl, pH 7.5, containing 2 mM EGTA, 2 mM EDTA, 1% Triton X-100, 0.5% Na–deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.2 mM PMSF, 10 µg/mL aprotinin, and 10 µg/mL leupeptin, and incubated for 30 min on ice. The lysate was centrifuged at 10000×g for 15 min at 4°C. The resultant supernatant was subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Immunoblot analysis was performed as described previously.16)
Tryptic Activity AssayMC9 cells were lysed in PBS containing 2 M NaCl and 0.5% Triton X-100 for 30 min on ice. The lysate was centrifuged at 10000×g fro 30 min at 4°C. Activity of tryptase in the lysate was measured using S-2288 as described previously.4)
We first confirmed the expression profiles of Gfi1 and Gfi1b in BMMCs during the co-culture period. The expression levels of Gfi1 were transiently increased on Day-4, whereas those of Gfi1b diminished over time, indicating the dynamic changes in expression of Gfi1 and Gfi1b during the co-culture period (Fig. 1A). Maturation of mast cells is usually evaluated by their granule maturation, such as histamine storage and mast cell-specific granule protease expression. Histamine synthesis, which is mediated by L-histidine decarboxylase (HDC), was induced by SCF in the presence or absence of Swiss 3T3 fibroblasts (Figs. 1B, C), whereas tryptic activity was significantly augmented only in the presence of SCF and Swiss 3T3 cells (Fig. 1D). Enzyme activities of chymase and carboxypeptidase A were undetectable in MC9 cells under all conditions investigated (data not shown). We then investigated the expression profiles of Gfi1 and Gfi1b in MC9/mock cells. Since tyrosine kinase, Lyn, is not expressed in Swiss 3T3 cells (data not shown), the amount of Lyn was measured as the loading control. We also confirmed that Gfi1 and Gfi1b were not expressed in Swiss 3T3 cells (data not shown). MC9/mock cells expressed both Gfi1 and Gfi1b under steady state conditions. SCF alone did not affect the expression levels of Gfi1 but down-regulated Gfi1b (Fig. 1E). Co-culture with Swiss 3T3 cells transiently down-regulated Gfi1 and abolished the expression of Gfi1b on Day-2. These results suggest that histamine synthesis should be independent of the expression Gfi1 and induction of tryptic activity may be prevented by Gfi1b.
(A) IL-3-dependent bone marrow-derived cultured mast cells were co-cultured with Swiss 3T3 fibroblasts in the presence of SCF for the periods indicated. Expression levels of Gfi1 and Gfi1b were determined by immunoblot analyses. Expression levels of β-actin were measured as the loading control. (B–D) MC9/mock cells were co-cultured with or without Swiss 3T3 fibroblasts in the presence of SCF (30 ng/mL) for 1 or 2 d. The cells were harvested and histamine content (B), HDC activity (C) and tryptic activity (D) were measured. The values were represented as the means±S.E.M. (n=3). (E) Expression levels of Gfi1 and Gfi1b in the cells were determined by immunoblot analyses and densitometrically measured. Expression levels of Lyn were measured as the loading control. The relative expression levels were presented as the means±S.E.M. (n=3). The values * p<0.05 are regarded as significant by one-way ANOVA with Dunnett multiple comparison test (vs. Day-0).
We then investigated the effects of stable expression of Gfi1 and Gfi1b, which was performed using the recombinant lentivirus, on tryptase expression and histamine synthesis. We confirmed that the lentivirally-transduced genes were expressed in MC9 cells by immunoblot analysis (Figs. 2A, B). The amount of Gfi1 was not significantly changed in the cells with the transgenes, whereas that of Gfi1b was increased in the cells expressing the exogenous Gfi1b transgene. It might be unlikely that lentiviral expression of Gfi1 was unsuccessful in this system because the expression levels of Gfi1 were significantly higher in the cells harboring the transgene under the co-culture condition (Fig. 3A). Gfi1 and Gfi1b were found to repress their own transcription and Gfi1b was reported to repress Gfi1 transcription in T cells.17–19) Exogenously-transduced Gfi1 and Gfi1b proteins may regulate the endogenous expression of Gfi1 and Gfi1b. We then investigated histamine synthesis and tryptase expression in these cells with the transgenes and found no significant changes (Figs. 2C–E), indicating that augmented expression of Gfi1b alone could not affect histamine synthesis and tryptase expression.
Expression of Gfi1 and Gfi1b were enhanced in MC9 cells using the recombinant lentivirus encoding Gfi1 and Gfi1b cDNA (Gfi1, MC9/Gfi1 and Gfi1b, MC9/Gfi1b). (A, B) Expression levels of Gfi1 and Gfi1b were determined by immunoblot analyses and densitometrically measured. Expression levels of β-actin were measured as the loading control. The relative expression levels were presented as the means±S.E.M. (n=3). The values * p<0.05 are regarded as significant by one-way ANOVA with Dunnett multiple comparison test (vs. mock). These cells were characterized by measuring histamine content (C), HDC activity (D) and tryptic activity (E). The values were represented as the means±S.E.M. (n=3).
MC9 cells without or with lentivirally-transduced Gfi1 and Gfi1b genes (mock, MC9/mock, Gfi1, MC9/Gfi1, and Gfi1b, MC9/Gfi1b) were cultured with or without Swiss 3T3 cells in the presence of SCF (30 g/mL) for 2 d. (A) Expression levels of Gfi1 and Gfi1b in the cells was determined by immunoblot analyses and densitometrically measured. The relative expression levels were presented as the means±S.E.M. (n=3). The values * p<0.05 are regarded as significant by one-way ANOVA with Tukey–Kramer multiple comparison test. Expression levels of Lyn were measured as the loading control. These cells were characterized by measuring histamine content (B), HDC activity (C) and tryptic activity (D). The values were represented as the means±S.E.M. (n=3). The values * p<0.05 and # p<0.05 are regarded as significant by one-way ANOVA with Tukey–Kramer multiple comparison test.
SCF and co-culture with Swiss 3T3 cells were found to affect the expression levels of Gfi1 and Gfi1b (Fig. 1E), raising the possibility that stable expression of Gfi1 and Gfi1b may alter the phenotypic changes observed during the co-culture period. Forced expression of Gfi1 resulted in enhancement of total Gfi1 expression in MC9 cells in the presence or absence of Swiss 3T3 cells (Fig. 3A). On the other hand, forced expression of Gfi1b augmented total expression of Gfi1b. The profiles of histamine content under three different culture conditions were not affected by the transgenes (Fig. 3B), whereas HDC activity was significantly up-regulated in the cells expressing the Gfi1b transgene in the presence of Swiss 3T3 cells, indicating the positive roles of Gfi1b in gene expression of HDC (Fig. 3C). Relationship between tryptase expression and Gfi1/Gfi1b expression was found to be more complicated. Induction of tryptase expression during the co-culture period was significantly suppressed in both kinds of MC9 cells expressing the Gfi1 transgene and the Gfi1b transgene (Fig. 3D), indicating that one-sided expression of Gfi1/Gfi1b suppressed the induction of tryptase. In accord with this hypothesis, comparable expression of Gfi1 and Gfi1b, which was observed in MC9 cells expressing the Gfi1b transgene in the presence of SCF alone and in MC9 cells expressing the mock transgene co-cultured with Swiss 3T3 cells, did not prevent the induction of tryptase.
Gfi1 and Gfi1b are involved in regulation of growth and differentiation of various hematopoietic lineages, such as B cells, T cells, neutrophils, dendritic cells, macrophages, and erythrocytes. Here we add mast cells in this list. Gfi1 and Gfi1b have been identified as transcription repressors. For instance, in neutrophils, Gfi1 represses transcription of neutrophil elastase-2 (ELA2), and matrix metalloproteinase-8 (MMP8) by binding to their promoter regions.11,20,21) Since we previously demonstrated that activated murine neutrophils express HDC,22) it is possible that Gfi1 controls gene expression of HDC in neutrophils. Gfi1 and Gfi1b were found to share with the same consensus DNA sequence, which consists of 12-base-pairs TAAATCAC(A/T)GCA with a absolute requirement of AATC.23,24) The possible candidate sites for Gfi1/Gfi1b binding were found in 5′-upstream region of the Hdc gene and the Mcpt6 gene, which encodes a tryptase, mouse mast cell protease-6; −123 (5′-CAA ATC AGA AAG-3′) and −861 (5′-GAA ATC ATA TTC-3′) from the transcription initiation site of the Hdc gene, and −114 (5′-TAA ATC ACT ATT-3′) and −998 (5′-AAA ATC AAC TGA-3′) from that of the Mcpt6 gene. Gfi1 and Gfi1b may bind to these sites and modulate the transcription of the Hdc and Mcpt6 gene.
Gfi1 was found to enhance STAT3-mediated signaling in T cells by directly interacting with PAIS3, which is an inhibitor of STAT3.25) In mast cells, PIAS3 was reported to interact with a transcription factor MITF and to suppress its functions.26–28) Since MITF is one of the primary regulators of mast cell differentiation and regulates expression of granule proteases including tryptases,29) modulation of tryptase expression through Gfi1b may involve MITF in MC9 cells. In summary, we demonstrated that both Gfi1 and Gfi1b might be involved in the process of maturation of murine mast cells.
This study was supported in part by JSPS KAKENHI (Grant Number 23590077).
