Published online before print May 22, 2003
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* Laboratories of Nutrition and
Molecular Biology, Azabu University School of Veterinary Medicine, and
Azabu University Research Institute of Biosciences, Sagamihara, Japan; and
Laboratory of Cellular Biochemistry, RIKEN, Wako, Japan
Correspondence: Masayuki Funaba, Ph.D., Laboratory of Nutrition, Azabu University School of Veterinary Medicine, 1-17-71 Fuchinobe, Sagamihara 229-8501, Japan. E-mail: funaba{at}azabu-u.ac.jp
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Key Words: mouse mast cell protease-1 • transforming growth factor-ß • dose-dependent activity • immune response
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RI) provide the trigger for activation of mast cells, leading to secretion of granules containing histamine, leukotrienes, cytokines, and proteases [4
]. Although several local growth and differentiation factors positively and negatively affect the function of mast cells [5
], comprehensive understanding of cell growth, migration, differentiation, and activation of mast cells at the molecular level is not yet complete. Activins, dimers of inhibin/activin ß subunits, are members of the transforming growth factor (TGF)-ß superfamily. Similar to the other members of the TGF-ß family such as TGF-ßs and bone morphogenetic proteins (BMPs), activin signaling is initiated by the heteromeric cell-surface receptors composed of type I and type II receptor subunits. Each subunit is a transmembrane protein that is endowed with a cytoplasmic serine kinase domain [6 ]. Activin binds directly to the extracellular domain of the type II receptor, followed by recruitment of the type I receptor. The type II receptor kinase then phosphorylates the cytoplasmic regulatory sequence of the type I receptor, termed the GS domain [7 ]. This phosphorylation activates the type I receptor kinase, followed by phosphorylation of Smad2 and -3 at the C-terminal serines. Phosphorylated Smad2 and -3 oligomerize with Smad4 and translocate to the nucleus [8 9 10 11 ]. Type II receptors of activins partly overlap with those of BMPs, and signaling of activins and TGF-ßs is indistinguishable at the Smad level, except for activin receptor-like kinase 1 (ALK1)-mediated Smad signaling of TGF-ßs in endothelial cells [8 9 10 11] ( Table 1 ).
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Table 1. Signaling Molecules of Mammalian TGF-ß Superfamily1
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-minimum essential medium (-MEM) with 10% fetal bovine serum (FBS) and 10% pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) [18
, 19
]. More than 95% of the trypan blue-excluding viable cells were mast cells on the basis of staining with acid toluidine. P-815 cells, a mouse mastocytoma cell line, were cultured in RPMI 1640 with 10% FBS. IC-2 cells, a mouse mast cell progenitor cell line, were cultured in RPMI 1640 with 10% FBS and 10% PWM-SCM. Recombinant human activin A was kindly provided by Dr. A. F. Parlow through the National Pituitary and Hormone Distribution Program at The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; NIH, Bethesda, MD). Purified TGF-ß1 was purchased from Becton Dickinson (San Jose, CA). Recombinant mouse interleukin (IL)-3, IL-9, and stem cell factor (SCF) were from PeproTech EC (London, UK). PWM, phorbol 12-myristate 13-acetate (PMA) was from Seikagaku Kogyo (Tokyo, Japan), and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was from Sigma Chemical Co. (St. Louis, MO).
RNA isolation, reverse transcriptase-polymerase chain reaction (RT-PCR), and competitive RT-PCR
Total RNA from mast cells was isolated using an RNA isolation kit (RNeasy, Qiagen, Tokyo, Japan). RNA treated with DNase I (Gibco-BRL, Gaithersburg, MD) was reverse-transcribed and subjected to PCR as described previously [13
, 17
]. Primers to detect ligands and receptors for the TGF-ß family and Smads cDNAs were described previously [20
]. The cDNA from mouse ovary and RT product without treatment with RT were simultaneously subjected to PCR as a positive and negative control, respectively [13
, 17
, 20
]. To examine mouse mast cell protease-1 (mMCP-1) gene transcript (accession number NM008570) quantitatively, nucleic acid sequences coding positions 288–312 and 577–602 were used for the sense and antisense PCR primers, respectively. A competitor for mMCP-1 was a deletional mutation of the mMCP-1 PCR product, and a deleting cDNA segment was positions 313–425. The competitor was made by overlap-extension PCR of the native PCR product, followed by purification using the Suprec-02 column (Takara, Tokyo, Japan). The PCR primers and competitor for glyceraldehyde 3-phosphate dehydrogenase (G3PDH) are as described previously [13
]. A constant amount (5x10-5 atto mol for mMCP-1 and 5x10-4 atto mol for G3PDH) of the competitor template was coamplified with the specific primers with reverse-transcribed samples or varying amounts of the target cDNA standard in a 10-µl scale of PCR. The PCR products were separated on 2% agarose gels in 1 x Tris-acetate-EDTA buffer and visualized with ethidium bromide. The band intensity was quantified using densitometric analysis by Scion image (Frederick, MD). Quantification of the mRNA level in the sample was conducted as described previously [13
, 17
].
MTT assay
BMCMCs were seeded in triplicates in 96-well plates at 2.5 x 105 cells/50 µl in 10 ng/ml IL-3 in -MEM medium with the indicated concentrations of activin A or TGF-ß1. After 5 days of culture, 5 µl 5 mg/ml MTT was added for 4 h, followed by cell lysis and color development with 50 µl 0.04 M HCl in 2-propanol. Plates were analyzed in an enzyme-linked immunosorbent assay plate reader at 570 nm with a reference wavelength of 655 nm [21
].
DNA fragmentation assay
For DNA fragmentation assays, BMCMCs (5x106 cells/ml) were treated with or without activin A (2 nM) or TGF-ß1 (200 PM) for 72 h. BMCMCs were also treated with or without lipopolysaccharides (LPS; 10 µg/ml). Cells were lysed in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.2% Triton X-100). DNA from the cell lysates was extracted with phenol/chloroform and was precipitated with ethanol. DNA fragmentation was analyzed by electrophoresis on a 2% agarose gel [19
, 22
].
Lactate dehydrogenase (LDH) assay
To measure LDH activity in culture supernatant, BMCMCs (5x106 cells/ml) were treated with or without activin A (2 nM) or TGF-ß1 (200 PM). BMCMCs were also treated with or without LPS (10 µg/ml). At 72 h of culture, culture supernatants were collected, and LDH activity was measured by the method of Storrie and Madden [23
].
Morphological analysis
BMCMCs (2x106 cells/ml) were cultured with 10% PWM-SCM with or without activin A (2 nM) or TGF-ß1 (200 PM). At 24, 72, and 120 h of culture, BMCMCs were cytocentrifuged onto clean glass slides for 6 min at 1000 rpm (Cytospin3, Shandon, Runcorn, UK). Cytosmears fixed in Carnoy’s were stained with toluidine blue (pH 4.1). For all cytosmears, 500 or more BMCMCs were examined. Total cell number and the number of cells containing strongly metachromatic granules were counted, and the percentage of the metachromatic cells to total cells was calculated. For BMCMCs cultured for 72 h, a nuclear-cytoplasmic ratio was also calculated as the total area of nucleus divided by the area of cytoplasm in a cell with an imaging analyzer (MacScope analyzer, Mitani, Fukui, Japan).
Measurement of gene transcripts of mMCP-1
BMCMCs (2x106 cells/ml) were cultured with 10% PWM-SCM with or without the indicated concentration of activin A or TGF-ß1. In addition, BMCMCs cultured with 10% PWM-SCM were also cocultured with or without SCF (50 ng/ml) and IL-9 (10 ng/ml), and effects of activin A (2 nM) and TGF-ß1 (200 PM) were examined. At 48 h after treatment, BMCMCs were collected, and gene transcripts of mMCP-1 were determined by competitive RT-PCR as described above.
Migration assay
Migration assays were conducted as described in the previous studies [24
25
26
]. They were performed in 24-well dishes by use of micropore filter cups with a pore size of 8 µm (150-µm thickness nitrocellulose; Millipore, Bedford, MA), which were coated overnight with human plasma fibronectin at a concentration of 10 µg/ml at room temperature and air dried for at least 60 min before use. The indicated concentrations of activin A or TGF-ß1 (500 µl) were added to each well below the filter and 500 µl cell suspension (1x106 cells/ml) above the filter. Cells were allowed to migrate for 180 min at 37°C in 5% CO2. Nonadherent cells at the upper surface of the filter were washed twice with phosphate-buffered saline. The filter was fixed with methanol for 5 min, followed by staining with toluidine blue. The number of BMCMCs at the lower surface of the filter was counted under light microscopy. The migration of cells suspended in -MEM medium and with the same medium below the filter served as a control and was referred to as 100% migration [26
].
Gelatin zymography
BMCMCs (5x106 cells/ml) were incubated in serum-free -MEM medium with or without activin A (2 nM) or TGF-ß1 (200 PM). BMCMCs were also treated with or without PMA (50 nM). The culture supernatants were harvested at 72 h of culture. Gelatin zymography was conducted as described previously [13
, 19
]. For inhibition studies, gel slices were incubated overnight at 37°C in the presence of 20 mM EDTA. Gelatinolytic bands completely disappeared by the presence of 20 mM EDTA.
Statistical analysis
Data were presented as the mean ± SD. Comparisons between the group treated without activin A and TGF-ß1 and the treated group were conducted by Student’s t-test. For all analyses, P < 0.05 was considered significant.
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Table 2. Gene Expression of Molecules Involved in Signaling of the TGF-ß Superfamily in Mast Cells
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Activin effects on mast cell functions
RT-PCR analyses suggested that BMCMCs could be target cells of activins. Therefore, we evaluated the effects of exogenous activin A and structurally related TGF-ß1 on mast cell functions in BMCMCs. Although at present, no information is available on the in vivo concentration of activin A at sites of inflammation, the in vitro study revealed that activated macrophages secreted activin A at 2 nM [13
]. In addition, activated monocytes also secreted activin A at >3 nM [15
]. Furthermore, serum activin A concentrations of healthy men and women are 
20 pM [12
]. Therefore, in the present study, activin A was treated at the concentration of 0–4 nM. We examined effects of activin A on growth inhibition, apoptosis, morphological changes, gene expression of mMCP-1, migration, and gelatinolytic activities in BMCMCs.
MTT assays revealed that activin A inhibited MTT uptake in a dose-dependent manner, and the maximal inhibition of MTT uptake was detected in BMCMCs treated with 4 nM activin A; the IC50 concentration was estimated to be 2.1 nM (Fig. 1A , right). Treatment with TGF-ß1 also decreased MTT uptake in a dose-dependent manner (Fig. 1A , left). Growth inhibitory activity of TGF-ß1 was more potent, and the IC50 concentration was 185 pM.
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Figure 1. Activin A and TGF-ß1 inhibit cell growth but do not induce apoptosis in BMCMCs. (A) Growth inhibition of BMCMCs was evaluated in MTT assays as described in Materials and Methods. The data are expressed as the mean ± SD (n=3). Conc., Concentrations. (B) Effects of activin A (2 nM) and TGF-ß1 (200 pM) on DNA fragmentation were examined in unstimulated BMCMCs and LPS (5 mg/ml)-induced apoptotic BMCMCs. In addition, effects of LPS (0, 2.5, 5, and 10 mg/ml) were also examined. (C) LDH activity in culture supernatant of BMCMCs treated with or without activin A (2 nM), TGF-ß1 (200 pM), or LPS (10 mg/ml) was measured as described in Materials and Methods.
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Morphological analyses revealed that there were substantial differences between groups. BMCMCs without activin A and TGF-ß1 had pseudopodia, and the granules were less distinct. Treatment with 200 pM TGF-ß1 increased the number of BMCMCs containing strongly metachromatic granules (Fig. 2A and 2B >). In addition, more rounded and compact BMCMCs were detected (Fig. 2A ). The reduction in nuclear size was greater than that in cytoplasmic size, resulting in a decrease in the nuclear-cytoplasmic ratio (Fig. 2C) . Similar to TGF-ß1 treatment, 2 nM activin A increased the number of BMCMCs with metachromatic granules (Fig. 2A and 2B) . Activin A treatment was also associated with more rounded and compact BMCMCs, but the compact BMCMCs were clearly less than those treated with TGF-ß1 (Fig. 2A) . The nuclear-cytoplasmic ratio of BMCMCs treated with activin A exhibited intermediate value between control cells and cells treated with TGF-ß1 (Fig. 2C) .
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Figure 2. Activin A and TGF-ß1 change morphology of BMCMCs, which were cultured with 10% PWM-SCM with or without activin A (2 nM) or TGF-ß1 (200 pM). Cytosmears were stained with toluidine blue (pH 4.1). (A) Representative cytosmears cultured for 72 h are shown. (B) The percentage of cells containing strongly metachromatic granules to total cells is shown. The data are expressed as the mean ± SD (n=3). * and **, P < 0.05 and P < 0.01, respectively. (C) The nuclear-cytoplasmic ratio is shown. The data are expressed as the mean ± SD (n=3). *, P< 0.05.
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Figure 3. Activin A and TGF-ß1 increase mMCP-1 gene transcript in BMCMCs. Effects of activin A and TGF-ß1 on gene transcripts of mMCP-1 were examined by competitive RT-PCR in BMCMCs, which were cultured with 10% PWM-SCM with the indicated concentrations of activin A (C and D) or TGF-ß1 (A and B) for 48 h. (A and C) PCR using cDNA as a template was performed in the presence of a constant amount of competitor. A representative agarose gel electrophoresis of PCR products is shown. (B and D) The mRNA level of mMCP-1 was expressed as a ratio to G3PDH, the mRNA level without activin A and TGF-ß1 being set to 1. A representative result from two independent experiments is shown. The data are expressed as the mean ± SD (n=3). **, P < 0.01.
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Figure 4. IL-9 but not SCF enhances activin A- and TGF-ß1-induced gene expression of mMCP-1. The effects of activin A (2 nM) and TGF-ß1 (200 pM) on gene transcripts of mMCP-1 were examined by competitive RT-PCR in BMCMCs, which were cultured with 10% PWM-SCM with or without SCF (50 ng/ml) and IL-9 (10 ng/ml) for 48 h. (A) PCR using cDNA as a template was performed in the presence of a constant amount of competitor. A representative agarose gel electrophoresis of PCR products is shown. (B) The mRNA level of mMCP-1 was expressed as a ratio to G3PDH, the mRNA level without activin A, TGF-ß1, SCF, and IL-9 being set to 1. A representative result from three independent experiments is shown. The data are expressed as the mean ± SD (n=3). **, P < 0.01.
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100 fM, which was 2000 times lower concentration than the IC50 concentration for cell growth inhibition (Fig. 1A , left). Activin A also stimulated migration of BMCMCs (Fig. 5B) , although it did not have a migratory effect in human mast cells (HMCs) from umbilical cord blood and line HMC-1 [25
]. The reason for the inconsistent results is not clear, but it is possible that these HMCs might not express activin receptors. At 10 pM activin A, the number of migrating BMCMCs was the highest, and further increasing concentration decreased the number of migrating BMCMCs. The optimal concentration of activin A to induce migration of BMCMCs was 200 times lower than the IC50 concentration for cell growth inhibition (Fig. 1A
, right).
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Figure 5. Activin A and TGF-ß1 have an activity to induce migration in BMCMCs. Migration assays were performed in BMCMCs treated with the indicated concentrations of activin A (B) or TGF-ß1 (A) as described in Materials and Methods. A representative result from three independent experiments is shown. The data are expressed as the mean ± SD (n=3). Conc., Concentrations.
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Figure 6. Neither activin A nor TGF-ß1 affects secretion of gelatinase in BMCMCs. Effects of activin A (2 nM) and TGF-ß1 (200 pM) on activity of MMP-2 and MMP-9 were examined in culture supernatant of BMCMCs by gelatin zymography as described in Materials and Methods. A representative polyacrylamide gel stained with Coomassie brilliant blue R-250 is shown (A), and the relative band intensity of pro-MMP-9 is shown (B). MW, Molecular weight.
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RI and to increasing cytosolic Ca2+ concentrations, leading to secretion of activin A protein [17
]. In addition, up-regulation of activin A expression in response to the activation was also observed in peritoneal macrophages [13
] and monocytes [15
, 16
]. Therefore, activin A secreted from these activated immune cells may form a concentration gradient. It is possible that initially, activin A acts as a chemoattractant of circulating mast cell progenitors and that the migrated progenitors subsequently differentiate into mature mast cells with the increasing concentrations of activin A. The activation-induced activin A expression and subsequent migration and differentiation of mast cells suggest that activin A positively regulates the functions of mast cells as effector cells in the immune system. The function of activin A in BMCMCs was qualitatively similar to TGF-ß1 on cell growth inhibition, apoptosis, induction of mMCP-1 gene, migration, and gelatinase activity. Smad proteins mediate signaling by these proteins, although in some cases, the signals are also transmitted by Smad-independent mechanisms [32 ]. Activin signaling is distinct from TGF-ß signaling at the receptor level, but at present, no difference has been established between the intracellular signaling pathways of activin and TGF-ß at the Smad level [8 9 10 11] (Table 1) . Thus, these TGF-ß-like activities of activin A in BMCMCs suggest that a Smad-dependent pathway elicited these functions.
Irrespective of the qualitative similarity between the functions of activin A and TGF-ß1 in BMCMCs, activin A effects were quantitatively different from TGF-ß1 effects. The concentration of activin A that elicited cell growth inhibition was higher than that of TGF-ß1. The reason why activin A was a less potent inhibitor of cell growth is currently unknown. Similar results have been obtained in other cells, including B lymphocytes [33 34 35 36 ], ovary cells [37 ], Leydig cells [37 ], and liver cells [38 ].
The present results revealed that activin A and TGF-ß1 had an activity to induce gene expression of mMCP-1 in BMCMCs at nanomolar and picomolar ranges, respectively. The concentrations that increased the gene expression were comparable with those that inhibited cell growth for both ligands. In view of the inverse relationship among cell proliferation and differentiation, the limited localization of mMCP-1 in mature mucosal mast cells [27 ], and morphological characteristics [31 ], our data suggest that activin A as well as TGF-ß1 act as differentiation factors of mast cell progenitors. This stimulatory activity on differentiation was enhanced by IL-9 but not SCF; both are known to promote growth and development of mast cells [3 , 5 ]. At present, the molecular basis of the synergism of IL-9 and activin A or TGF-ß1 on induction of mMCP-1 gene is not known, but it is likely that Smad proteins act cooperatively with signal transducer and activator of transcription (STAT) proteins, which are signal mediators of IL-9 [39 ]. Cooperation of Smad and STAT on transcriptional activation of the vasoactive intestinal peptide gene has been reported [40 ].
Previous studies revealed maximal migratory responses to TGF-ß1 at femtomolar concentrations in mast cells [24 25 26 ], and the optimal concentration was 1000 times lower than the IC50 concentration to inhibit cell growth [25 ]. In view of similar effects of activin A to those of TGF-ß in several biological assays as revealed in this study and in the other studies [38 , 41 42 43 ], we expected that activin A would also have an activity to induce migration of BMCMCs. However, we could not predict whether the concentration to elicit maximal migratory response would be in the femtomolar range or 1000 times lower than the IC50 concentration to inhibit cell growth, as activin A was less potent than TGF-ß1 on cell growth inhibition. Migration assays revealed that activin A also has a migration-inducing activity but only at a limited range of concentrations. However, as compared with TGF-ß1, the optimal concentration of activin A was higher, and the difference between the concentration to induce migration of BMCMCs and that to inhibit cell growth was smaller in activin A; the concentration of activin A was 10 pM, which was 200 times less than the IC50 concentration to inhibit cell growth, whereas that for TGF-ß1 was 100 fM and 2000 times lower. These results suggest that BMCMCs sense different concentrations of the same ligand, as well as concentration of the different ligands. In addition, it is suggested that BMCMCs respond to a concentration gradient of activin A more sensitively than TGF-ß1. This concentration-dependent function of activin A is also known in Xenopus blastula cells. Activin had the ability to switch animal cap cells of a blastula from their normal epidermal or neural fate into mesoderm cell types, ranging from blood, through muscle, to notochord, by activation of different genes as the activin concentration increased [44 ]. Among genes induced by activin A, Xbrachyury and Xgoosecoid genes were activated by the ability of cells to sense ligand concentration by the absolute number of occupied receptors per cell; 100 and 300 molecules of bound activin induced Xbrachyury and Xgoosecoid genes, respectively [45 ]. A threefold difference in the number of occupied activin receptors was maintained in the nuclear Smad2 protein level to activate the genes [46 ].
When circulating mast cell progenitors are to infiltrate the sites of inflammation, they must degrade extracellular matrix, which is partly achieved by release of MMPs. Previous studies revealed that MMP-2 and -9, which have gelatinase activity, were produced in mast cells [30 , 47 , 48 ] and that MMP-2 activity was increased by activin A treatment in mouse peritoneal macrophages [13 ]. Therefore, we examined whether the activity of these enzymes can be modulated by activin A and TGF-ß1. Our results on gelatin zymography indicated that neither activin A nor TGF-ß1 affected the activities of MMP-2 and -9 in BMCMCs. These results suggest that local factors other than activin A and TGF-ß1 regulate expression and activity of MMPs if these enzymes are involved in the infiltration of mast cell progenitors. Several cytokines and bacterial constituents are reported to affect MMP-9 activity in mast cells [19 , 30 , 48 ].
In summary, the present results demonstrate that activin A inhibits cell growth, induces terminal differentiation, and acts as a chemoattractant in mast cell progenitors. Previous studies indicated the up-regulation of activin A but not TGF-ß in response to the activation of immune cells including mast cells [17
], macrophages [13
], and monocytes [15
]. In addition, activin A was functionally different from TGF-ß in mouse peritoneal macrophages in the regulation of MMP [13
, 49
]. There may be different functions of activin A from TGF-ß also in mast cells, which were not identified in the present study. Thus, by a distinct mode from TGF-ß, activin A may play a role in the inflammatory process, possibly in concert with well-known inflammatory cytokines such as ILs, interferons, and tumor necrosis factor .
Received January 9, 2003; revised February 23, 2003; accepted February 26, 2003.
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RI: coupling form to function Adv. Immunol. 76,325-355[Medline] J. Biol. Chem. 275,30894-30900 J. Immunol. 167,4008-4016This article has been cited by other articles:
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