Published online before print October 2, 2003
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* Department of Pathology and Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque; and
Department of Laboratory Medicine, University of California, San Francisco
1Correspondence: Department of Pathology, University of New Mexico School of Medicine, CRF Rm. 205, 2325 Camino De Salud NE, Albuquerque, NM 87131. E-mail: valh{at}unm.edu
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Key Words: mast cells/basophils • signal transduction • apoptosis • cellular proliferation • cellular activation
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mast cell (MC) differentiation and proliferation are regulated by cytokines, principally interleukin-3 (IL-3) [7
] and the c-kit ligand, stem cell factor (SCF) [8
]. The murine IL-3 receptor (IL-3R) is composed of an 
-subunit and a ßc-chain subunit, which are phosphorylated on tyrosine residues upon ligand stimulation (reviewed in ref. [9
]). IL-3 binding leads to phosphorylation of ßc, the activation of the Janus family of protein kinase (JAK)2/signal transducer and activator of transcription (STAT)5, phosphatidylinositol-3 kinase (PI-3K), and Ras mitogen-activated protein kinase (MAPK) signaling pathways and to cell proliferation, survival, and differentiation. Similarly, the binding of SCF to the receptor tyrosine kinase, c-kit, stimulates protein phosphorylation and cell proliferation, survival, and differentiation.
It has been reported that Lyn deletion has no effect on [5
, 10
, 11
] or reduces [12
] the IL-3 and SCF-mediated proliferation and differentiation of mouse bone marrow-derived MC (BMMC) precursors in vitro and the numbers of mature MC in vivo. We were thus surprised to discover that Lyn-deficient mice have more peritoneal MC than wild-type (WT) mice. This observation led us to re-examine the role of Lyn in MC growth, death, and signaling pathways. We report that Lyn-/- BMMC divide faster in response to IL-3 and SCF during the 4–5 weeks required to produce a >95% pure population of granular, receptor with high affinity for IgE (Fc
RI)-positive BMMC. Furthermore, differentiated Lyn-/- BMMC continue to proliferate more rapidly than WT BMMC in cytokine-containing media, and they undergo less apoptosis when cytokines are removed. Additionally, Lyn-/- BMMC support greater phosphorylation of the prosurvival kinase, Akt, and the proliferative kinase, extracellular-regulated kinase (ERK)1/2, when stimulated with IL-3. Thus, Lyn behaves as a negative regulator of murine MC survival and proliferation.
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chain mAb antibody and isotype-control biotinylated rat anti-mouse IgG2a were from PharMingen (San Diego, CA). Mouse antiphospho-ERK1/2 and rabbit antibodies to phospho-STAT3 (Tyr 705), STAT3, phospho-STAT5a/b (Tyr 694), ERK1/2, phospho-c-Jun N-terminal kinase (JNK), JNK, phospho-Akt, and Akt were from Cell Signaling Technology (Beverly, MA). Rabbit antibodies to antiphospho-p38, p38, and STAT5a/b were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse Ig antibodies and goat anti-rabbit Ig antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Mouse recombinant (r)IL-3 and mouse rSCF were purchased from Biosource International (Camarillo, CA).
Cells and cell culture conditions
WT and Lyn knockout mice [4
] of the same background strain (C57BL6) were bred in specific, pathogen-free facilities in the University of New Mexico Animal Research Facility (Albuquerque, NM). BMMC were obtained by culturing BM from 8- to 12-week-old C57BL6 WT and Lyn-/- mice. Briefly, mice were killed, and BM were flushed from the femurs and tibias. The cells were cultured at a starting density of 0.5 x 106 cells/ml at 37°C in a humidified atmosphere containing 5% CO2 in RPMI-1640 medium, supplemented with 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 10% fetal calf serum (FCS; Hyclone Laboratories Inc., Logan, UT), 55 µM ß-2-mercaptoethanol (2-ME), 1 mM HEPES (complete RPMI medium), and 30% WEHI-3-conditioned medium, as a source of IL-3 [14
]. All cell-culture reagents were purchased from Life Technologies (Grand Island, NY). The nonadherent cells from the BM cultures were transferred every 7 days into fresh IL-3-containing medium. Cell cultures were maintained for 5–8 weeks, and toluidine blue staining identified MC. For in vitro growth curves, 8 x 106 BM cells per plate were cultured in complete RPMI with 30% WEHI-3-conditioned RPMI medium or in RPMI medium supplemented with combinations of various growth factors (increasing amounts of IL-3 or 10 ng/ml IL-3 plus 20 ng/ml SCF). Each culture was performed in duplicate using cells plated at identical densities (5x105 cells/ml). Weekly cell counts were recorded by exclusion of trypan blue. By 5 weeks, essentially 100% of cells contained the prominent granules characteristic of differentiated MC. The experiments described below were all performed with 5- to 8-week-old MC cultures.
Isolation and staining of mouse peritoneal MC
Killed mice were injected intraperitoneally with 1–2 ml Hanks’ buffer without calcium and magnesium. The cavity was gently massaged, and the peritoneal fluid was aspirated and placed on ice. Cells were centrifuged at 1000 rpm and washed twice with Hanks’ medium. A portion of the cells was fixed and stained using methanol and 0.06% toluidine blue at a ratio of 1:1:1 (cells:methanol:0.06% toluidine blue) for 5 min. Total cells (excluding occasional erythrocytes) and total MC (recognized by their characteristic toluidine blue-stained granules) were counted with a hemacytometer.
Analysis of c-kit, IL-3, and FcRI expression
c-Kit receptor and IL-3R densities were measured by incubating BMMC with 1 µg/ml biotinylated rat anti-mouse CD117 (c-kit) antibody or with 1 µg/ml biotinylated rat anti-mouse IL-3R chain antibody for 1 h at 4°C. Control cells were incubated with the isotype controls, 1 µg/ml biotinylated rat IgG2b, or 1 µg/ml biotinylated rat IgG2a for 1 h at 4°C. For IgE receptor density analysis, cells were incubated overnight with 1 µg/ml biotinylated anti-DNP IgE at 37°C. Cells were washed twice with phosphate-buffered saline (PBS) and reincubated with biotinylated anti-DNP IgE for 1 h at 4°C. All cells were washed twice with PBS and incubated with avidin-R-phycoerythrin (A-PE; Molecular Probes, Inc., Eugene, OR; 1:200) for 1 h at 4°C. As a control, cells that were not pretreated with IgE were stained with A-PE. Fluorescence was analyzed by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA).
Proliferation assays
BMMC were harvested, washed, and suspended in fresh medium with the original additives in the presence or absence of serum. The cells were divided into four equal portions and incubated for 24 or 48 h at 37°C in 5% CO2 in flat bottomed, 96-well microtiter plates at a density of 2 x 105 cells/0.2 ml per well. [3H]-Thymidine (Amersham, Arlington Heights, IL; 0.5 µCi) was added to each well 4 h before harvesting 24-h samples or 18 h before harvesting 48-h samples. Cells were harvested with an automated cell harvester and analyzed in a Beckman LS 1801 liquid scintillation counter (Beckman Instruments, Fullerton, CA). Results are reported as averages ± SEM. Data were graphed and analyzed with Graphpad Prism software.
Cell-cycle analysis
BMMC were harvested, washed, starved of growth factors and serum for 4 h, and cultured in the presence or absence of different growth factors at 5 x 105 cells/ml in complete RPMI for 24 or 48 h. Cells were harvested again and washed twice in PBS. Cell-cycle analysis was performed by staining with 1 ml Krishan buffer [15
] consisting of 0.1% sodium citrate, 0.3% (v/v) Nonidet P-40, 50 µg/ml propidium iodide (PI; Sigma Chemical Co., St. Louis, MO), and 25 µg/ml RNase A (Clontech, Palo Alto, CA), pH 7.4, at 4°C for 1 h. Cells were washed twice with PBS and resuspended in 0.5 ml Krishan buffer. Cell-cycle distribution was analyzed using a FACScan flow cytometer. The percentages of cells in G0 + G1, S, and G2 + M were calculated using Modfit software (Verity Software House, Topchan, ME).
Assays for caspase activity
Total caspase activity was measured with a CaspaTag Caspase activity kit (Intergen, Purchase, NY) according to the protocol supplied by the manufacturer. Briefly, BMMC were washed with RPMI medium and resuspended at 106 cells per ml in the absence or presence of various growth factors (RPMI medium supplemented with 30% WEHI-3-conditioned medium; 20 ng/ml SCF; 10 ng/ml IL-3; or 10 ng/ml IL-3 plus 20 ng/ml SCF). Samples were incubated with the carboxyfluorescein-labeled fluoromethyl ketone caspase-specific inhibitor (FAM-VAD-FMK) in a 5% CO2 incubator at 37°C for 60 min. The cells were washed, stained with PI, and analyzed by two-color flow cytometry using a FACScan flow cytometer. Caspase activity was measured from the fluorescence derived from the covalent binding of inhibitor to active caspases in the cells.
Cell lysates and immunoblotting
BMMC were incubated overnight in complete RPMI medium, then washed in RPMI medium, and activated with 30 ng/ml IL-3 for the indicated times. Resting and activated cells were lysed in ice-cold 1% Brij 96-containing lysis buffer (50 mM TrisHCl, pH 7.2, 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml antipain, 10 µg/ml leupeptin) on ice for 15 min. Lysates were clarified by centrifugation at 4°C for 15 min at 13,000 rpm. Solubilized proteins were resolved by 7.5, 10, or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose membranes, and blocked for 1 h by incubation with 3% Ig-free bovine serum albumin (Sigma Chemical Co.) for incubation with phospho-specific antibodies to selected signaling proteins (Akt, ERK1/2, p38, STAT5a/b, STAT3) or with 5% milk. Membranes were incubated for 2 h at room temperature with the indicated antibody, washed, and probed with HRP-conjugated goat anti-rabbit or goat anti-mouse IgG antibodies. Membranes were developed with SuperSignal substrate (Pierce) and exposed to film. The same blots were stripped with stripping buffer (62.5 mM Tris, pH 6.8, 100 mM 2-ME, 2% SDS) for 30 min at 60°C, washed, and reprobed with antibodies to the same signaling proteins to verify equal loading.
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View this table: [in a new window] |
Table 1. Lyn-/- Mice Have More Peritoneal MC Than WT Mice
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RI (Fig. 1B)
, and IL-3 (Fig. 1C)
. In measurements made at any time between 5 and 8 weeks of culture, the levels of cell-surface FcRI, c-kit, and IL-3R were always very similar between WT and Lyn-/- BMMC. However, total cell numbers were always higher for the Lyn-/- cultures (Fig. 2A
).
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Figure 1. Surface c-kit, FcRI, and IL-3R expression is the same in BMMC from WT and Lyn-/- mice. BM-derived cells were cultured for 6 weeks in 30% WEHI-3-conditioned medium. The cells were harvested, and c-kit and IL-3R were labeled with biotinylated specific antibody followed by A-PE. Control staining used an isotype-matched mAb and A-PE. FcRI were labeled with biotinylated monoclonal anti-DNP IgE Ab followed by A-PE. Control staining was with R-PE conjugate alone. % pos, Percent of positively stained cells for the indicated receptor; m. fl., mean fluorescence.
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Figure 2. Greater expansion of Lyn-/- BM-derived cells grown in medium supplemented with growth factors. BM-derived cells were cultured in 30% WEHI-3-conditioned medium (A); 3, 10, 30 ng/ml rIL-3 (B), or 10 ng/ml rIL-3 and 20 ng/ml rSCF (C). Media were changed weekly, and the numbers of cells excluding trypan blue were recorded over 5 weeks. Data represent the average of two separate experiments performed with duplicate cultures. *, **, ***: Values are significantly different (P<0.05, P<0.01 and P<0.005; Student’s t-test) from other values in the same set.
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Lyn-/- BMMC incorporate more thymidine and have a greater percentage of cells in the S-phase
To determine whether the increased numbers of BMMC cultured from the BM of Lyn-/- mice were a result of increased DNA synthesis or decreased cell death, thymidine incorporation, cell cycling, and apoptosis were measured in WT and Lyn-/- MC.
DNA synthesis was measured in newly differentiated 5- to 6-week-old WT and Lyn-/- BMMC. Lyn-/- MC incorporated more thymidine than WT MC when incubated for 24 or 48 h in RPMI medium supplemented with their original WEHI-3-conditioned medium or with IL-3 (Fig. 3A and 3B ). In medium supplemented with SCF alone, WT and Lyn-/- MC incorporated comparable amounts of thymidine at 24 h (Fig. 3A) , and Lyn-/- MC incorporated more thymidine at 48 h (Fig. 3B) . Conversely, in medium containing IL-3 and SCF, Lyn-/- BMMC incorporated more thymidine than WT BMMC at 24 h (Fig. 3A) , and both cell types incorporated comparable amounts of thymidine at 48 h (Fig. 3B) .
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Figure 3. Increased proliferation of Lyn-/- BMMC. BM-derived cells from WT and Lyn-/- mice were cultured for 5–6 weeks in 30% WEHI-3-conditioned medium to produce BMMC and were then transferred to media supplemented with growth factors [30% WEHI-3-conditioned medium, recombinant murine (rm)IL-3, rmSCF, or IL-3 plus SCF] and [3H]-thymidine for 24 h (A) or 48 h (B). (C) Proliferation was measured in 8-week-old BMMC stimulated with growth factors for 48 h. (D) BMMC (6-weeks old) were stimulated with growth factors for 24 h in the absence of FCS. Data are presented as mean ± SEM for two experiments done in quadruplicate for each condition. Mean values significantly different from WT levels are indicated: *, P< 0.05; **, P< 0.01; ***, P < 0.005, Student’s t-test.
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Withdrawal of FCS from the medium reduced total thymidine incorporation but provided the most dramatic demonstration of the greater proliferative capacity of the Lyn-/- BMMC (Fig. 3D) .
Cell-cycle analyses confirmed the faster growth rate of Lyn-/- BMMC. MC were cultured with the same growth factors used for thymidine incorporation studies, and the content of DNA was analyzed by flow cytometry following labeling of nuclei with PI. Lyn-/- MC had a significant increase in the proportion of cells in the S/G2/M phase of the cell cycle compared with WT BMMC when incubation was for 24 h in WEHI-3-conditioned RPMI medium (WT, 3±0.6%; Lyn-/-, 8.3±0.3%; mean±SEM, n=3; P<0.05); in RPMI with added IL-3 (WT, 5.0±0.6%; Lyn-/-, 11±1.5%; mean±SEM, n=3; P<0.01); and in RPMI with added IL-3 plus SCF (WT, 11.7±1.5%; Lyn-/- 27.7±0.3%; mean±SEM, n=3; P<0.001; Fig. 4 A ). Similarly, a higher percentage of Lyn-/- BMMC were in S/G2/M compared with WT BMMC when stimulation was for 48 h in RPMI supplemented with WEHI-3-conditioned medium (WT, 5.7±0.9%; Lyn-/-, 11.3%±1.2%; mean±SEM, n=3; P<0.01); with IL-3 (WT, 8.7±0.3%; Lyn-/-, 14.7±0.3%; mean±SEM, n=3; P<0.001); and with IL-3 plus SCF (WT, 26.0±0.6%; Lyn-/-, 33.3±0.9%; mean±SEM, n=3; P<0.001; Fig. 4B ). In RPMI supplemented with SCF alone, WT and Lyn-/- BMMC had comparable percentages of cells in the S/G2/M phase of the cell cycle at 24 h (WT, 21.5±1.5%; Lyn-/-, 27.2±0.8%; mean±SEM, n=3; P>0.05; Fig. 4A ). However, there was a significant increase in the proportion of Lyn-/- BMMC versus WT BMMC in S/G2/M after stimulation with SCF at 48 h (WT, 23±1.2%; Lyn-/-, 35.7±1.5%; mean±SEM, n=3; P<0.001; Fig. 4B ).
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Figure 4. Lyn-/- BMMC pass more rapidly through the cell cycle in response to growth-factor stimulation. (A) Six-week-old WT and Lyn-/- BMMC were suspended at 0.5 x 106 cells/ml in complete culture medium supplemented with 30% WEHI-3-conditioned medium, rIL-3 (10 ng/ml), or rSCF (20 ng/ml) for 24 h (A) or 48 h (B). Nuclei were labeled with PI, and the percentage of cells in (G0+G1), S, (G2+M) was determined by flow cytometry. Data are presented as mean ± SEM for three independent experiments. Mean values significantly different from WT levels are indicated: *, P< 0.05; **, P< 0.01; ***, P< 0.001, Student’s t-test.
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In Figure 5 A , WEHI-conditioned medium was replaced with RPMI medium alone, and the percentages of early (caspase+/PI-; lower right quadrants) and late (caspase+/PI+; upper right quandrants) apoptotic cells were quantified using flow cytometry. In the absence of growth factors, WT and Lyn-/- MC had equivalent numbers of early and late apoptotic cells after 24 h of growth-factor deprivation. However, a higher percentage of apoptotic WT MC was present at 48 h and at 72 h.
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Figure 5. Caspase activation induced by growth-factor deprivation is delayed in Lyn-/- BMMC. Six-week-old WT and Lyn-/- BMMC obtained from BM cultures grown in 30% WEHI-3-conditioned medium were cultured at a density of 1 x 106 cells/ml in the absence of growth factors for 24, 48, or 72 h (A) or in the presence of growth factors (B). Caspase activation was determined by fluorescence intensity derived from the caspase-bound inhibitor. Results shown are representative of seven independent experiments. Numbers (inset) report percent early apoptotic (caspase+/PI-; lower right quadrants) and late (caspase+/PI+; upper right quadrants) apoptotic. FITC, Fluorescein isothiocyanate.
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In Figure 5B , WT and Lyn-/- MC were maintained in 30% WEHI-3-conditioned medium or transferred to RPMI medium with growth factors (10 ng/ml IL-3, 20 ng/ml SCF, or 10 ng/ml IL-3+20 ng/ml SCF) for 24 and 48 h before performing the caspase activation assay. In this and six replicate experiments, the numbers of apoptotic cells in the presence of growth factors were essentially the same in WT and Lyn-/- MC. Thus, the greater numbers of Lyn-/- BMMC in the presence of growth factors result primarily from their more rapid proliferation and not from decreased apoptosis.
IL-3-induced phosphorylation of Akt and ERK1/2 is increased in Lyn-deficient MC
Activities of the survival kinases, Akt, and the proliferative MAPK family members, ERK1/2, stress-activated protein kinase (SAPK)/JNK, and p38 were measured by immunoblotting with antibodies to the phosphorylated (active) forms of these enzymes.
When cells were deprived of IL-3, Lyn-/- BMMC exhibit higher basal levels of phosphorylated Ser-473 (activated) Akt than WT BMMC (Fig. 6 A ). Stimulation with IL-3 induced rapid phosphorylation of Akt in WT and Lyn-/- BMMC (Fig. 6A) . However, phosphorylation of Akt was prolonged in Lyn-/- BMMC (Fig. 6A) . Thus, increased Akt activity is likely to contribute to the enhanced survival of Lyn-/- BMMC.
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Figure 6. Increased IL-3-mediated Akt and ERK1/2 activation in Lyn-/- BMMC. Six- to 8-week old WT and Lyn-/- BMMC grown in 30% WEHI-3-conditioned medium were incubated in complete RPMI medium overnight and stimulated with 30 ng/ml IL-3 for the indicated times. Proteins from total cell lysates from 5 x 105 cells/lane were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and analyzed by immunoblotting with phospho-specific antibodies to Akt (A), ERK1/2 and p38 (B), and STAT5a/b and STAT3 (C). Membranes were stripped and reprobed with antibodies to total protein to verify equal loading. Each experiment was performed at least three times.
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As STATs are targets in IL-3-mediated signaling and are involved in cell proliferation, we also analyzed the levels and phosphorylation of STAT3 and STAT5 in resting and IL-3-stimulated BMMC. The anti-STAT3 antibodies recognize Stat3 (92 kDa) and its splice variant Stat3ß (83 kDa), and the anti-STAT5 antibodies recognize STAT5a and STAT5b (
90 kDa). The levels and intensities of phosphorylation of STAT3 were similar at all time points between WT and Lyn-/- BMMC (Fig. 6C)
. The levels and phosphorylation of STAT5 were lower at all time points in Lyn-/- BMMC than in WT BMMC (Fig. 6C)
. These data fail to implicate STATs in the hyperproliferation of IL-3-treated Lyn-/- BMMC.
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Our data differ from previous studies by Nishizumi and Yamamoto [10
], Kawakami et al. [11
], and O’Laughlin-Bunner et al. [12
]. The Nishizumi and Kawakami groups [10
, 11
] reported that IL-3-stimulated Lyn-/- BM generates normal numbers of MC, and O’Laughlin-Bunner et al. [12
] reported that SCF-induced proliferation of BM progenitors and BMMC is impaired in Lyn-/- cells. It is likely that our difference with Nishizumi and Yamamoto [10
] and Kawakami et al. [11
] can be explained by their predominant use of newly differentiated MC. Both groups worked with 4-week-old cultures, and we see greater differences in rates of cell expansion beyond that point. Furthermore, we performed survival assays using a caspase assay that gives a more accurate measure of apoptosis than trypan blue exclusion [11
]. The difference between our data and those of O’Laughlin-Bunner et al. [12
], obtained using Lyn-/- mice from the same source [4
], is more puzzling. These investigators differentiated BMMC using IL-3-conditioned medium and then challenged the mature cells with high levels of SCF (100 ng/ml). Their phenotyping studies revealed greater levels of FcRI and c-kit expression on the Lyn-/- BMMC, whereas we see no significant difference between Lyn-/- and WT cells in FcRI, IL-3, and c-kit receptor expression. Similarly, Kawakami et al. [11
] and Parravicini et al. [16
] saw no difference in FcRI expression between WT and Lyn-/- BMMC. Their proliferation studies revealed less SCF-induced thymidine incorporation in Lyn-/- BMMC but with very low levels of thymidine incorporation overall in comparison with our robust measures of thymidine incorporation [12
]. Their cell-cycle studies revealed many fewer cycling cells in SCF-stimulated cultures of Lyn-/- BMMC, again the opposite of our results. As Lyn has a multitude of positive and negative regulatory roles in cell physiology, it is possible that subtle differences in the age or health of the donor mice or in culture conditions could result in very different cell growth characteristics.
Cell growth and survival are regulated by a wide variety of signaling pathways, including JAKs and their targets, STATs, the PI-3K/Akt pathway, and the MAPK pathways. Based on the higher phosphorylation of Akt and ERK1/2 in Lyn-/- BMMC, our data implicate the prosurvival PI-3K/Akt pathway and the proproliferative ERK1/2 MAPK pathways in their greater proliferation and resistance to apoptosis in comparison with WT BMMC. Given the diverse roles for Lyn in cell regulation, it would not be surprising if other cell-signaling pathways involved in promoting cell-cycle progression and preventing apoptosis in response to IL-3 are also up-regulated in Lyn-deficient cells. Conversely, we found no evidence for increased STAT3 or STAT5 phosphorylation in Lyn-/- BMMC. We also found no evidence for altered p38 or JNK activation between WT and Lyn-/- BMMC during a 24-h time course of growth-factor deprivation (data not shown) or after stimulation with IL-3. Thus, the absence of Lyn does not cause a nonspecific up-regulation of all pro-growth, antiapoptosis signaling pathways.
The mechanism(s) linking Lyn deficiency to increased MC proliferation remains to be defined. Several lines of evidence point to the Src homology 2-containing inositol phospholipid phosphatase (SHIP) as a possible, direct intermediate between the absence of Lyn and the occurrence of excess signaling through the PI-3K/Akt pathway and the ERK1/2 pathway. Thus, Lui et al. [17
] identified SHIP as a crucial negative regulator of growth-factor receptor-mediated Akt activation and myeloid cell survival. They also showed that neutrophils and BMMC from SHIP-/- mice are less susceptible to apoptosis induced by growth-factor withdrawal [17
]. Furthermore, increased ERK1/2 and Akt phosphorylation has been shown in SHIP-/- B cells [18
19
], and SHIP-/- BMMC stimulated via the high-affinity IgE receptor [20
] or with SCF [21
] have increased and prolonged ERK1/2 phosphorylation. Finally, our own preliminary studies indicate that SHIP activity is reduced in Lyn-/- BMMC in comparison with WT BMMC (V. Hernandez-Hansen et al., manuscript in preparation). However, other factors including differences in the expression of cytokines could also contribute to the increased proliferation of Lyn-/- BMMC. Kawakami et al. [11
] reported increased expression of IL-2 and tumor necrosis factor expression in Lyn-/- MC stimulated overnight with antigen. If IL-3 or SCF also induce higher levels of cytokine and growth-factor expression in Lyn-deficient than in WT cells, the result could be greater signaling to cell survival and proliferation via pathways that are indirectly regulated by Lyn. Further studies are underway to distinguish between direct and indirect roles for Lyn in the negative regulation of IL-3 and SCF signaling to MC survival and proliferation.
Highlighting the critical role of Lyn in hematopoietic regulation, aged, Lyn-deficient mice develop a dramatic increase in myeloid progenitors, splenomegaly, disseminated monocyte/macrophage tumors [3 ], and B cell lymphomas (our unpublished results). Further studies on the Lyn-mediated, negative regulation of cytokine-mediated BMMC proliferation may reveal mechanisms that predispose Lyn-deficient mice to myeloid expansion and tumor development. New insight into human oncogenesis may in turn emerge from this improved understanding of the consequences of deregulated Lyn activity in hematopoietic cells.
Received May 16, 2003; revised August 20, 2003; accepted August 21, 2003.
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RI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions J. Exp. Med. 185,663-672
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K. W. Harder, C. Quilici, E. Naik, M. Inglese, N. Kountouri, A. Turner, K. Zlatic, D. M. Tarlinton, and M. L. Hibbs Perturbed myelo/erythropoiesis in Lyn-deficient mice is similar to that in mice lacking the inhibitory phosphatases SHP-1 and SHIP-1 Blood, December 15, 2004; 104(13): 3901 - 3910. [Abstract] [Full Text] [PDF]
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