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Department of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Multiple transcription factors are activated in the cytoplasm in response to growth factors and then shuttled to the nucleus where they exert positive or negative control over cellular genes. Generally, the constant exchange of macromolecules between the nucleus and the cytoplasm occurs through the nuclear pore complex (4). These transport processes are regulated by mechanisms in which molecules are restricted to a particular compartment until specific signals stimulate their migration. The major pathway of nuclear protein import is mediated by nuclear localization signal (NLS) sequences that are characterized by one or more clusters of basic amino acids (4). USF2 is one of the few transcription factors that contain two domains capable of mediating its translocation to the nucleus (5). In addition to the conventional basic NLS region, it contains a highly conserved USF-specific region that is involved in its nuclear translocation. The function of this USF-specific NLS was further investigated in the present work.
Cytokines and lymphokines have a vital influence on the regulation, maturation, activation, proliferation, and specific functions of mast cells. Among the predominant cytokines that regulate the function of murine mast cells is IL-3. IL-3 is a T cell-derived growth factor that has been extensively characterized in both mouse and man (6). It is known to stimulate the proliferation and differentiation of a broad spectrum of hemopoietic cells, including pluripotential stem cells, mature megakaryocytes, macrophages, and mouse mast cells.
In recent years rapid nuclear translocation of transcription factors has been found to be associated with cell activation by external stimuli (7). Here we provide evidence of the role of USF2 in the survival of IL-3-dependent cells, specifically murine mast cells, indicating a function of USF2 in cell growth. This was achieved by demonstrating IL-3 receptor-mediated movement of USF2 from the mast cell cytosol to the nucleus and the role of this nuclear translocation in mast cell survival.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MC-9 cells obtained from the American Type Culture Collection (ATCC, Manassas, VA) were maintained at 37°C in RPMI 1640 medium supplemented with 2 mM L-glutamine, 2 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD), 50 µM b-mercaptoethanol (Fisher Scientific, Medford, MA), 10% FCS (Bio-Lab, Jerusalem, Israel) (growth medium), and the lymphokines IL-3 and IL-4 (kindly provided by I. Clark-Lewis, Vancouver, Canada) (3). Bone marrow-derived mast cells (BMMC) were developed in culture from bone marrow cells derived from C57BL mice (8) and grown and maintained at 37°C in growth medium supplemented with IL-3 and IL-4.
Cell activation
MC-9 cells or BMMCs were washed and preincubated in RPMI 1640
medium alone for 60 min and then incubated with IL-3 for various
periods of time. Alternatively, IgE (3) was added to the medium for the
initial 60 min, and the cells were washed to remove excess IgE and then
resuspended in DNP-BSA (Ag)-containing medium and incubated for 45 min.
The IgE used was directed against DNP hapten coupled to BSA. This
is a multihapten Ag that is useful for coupling of the type I receptors
for IgE (Fc
RI). Some samples were exposed to a sublethal dose (0.35
µg/ml) of calcium ionophore (A23187), which was added to washed cells
that were then incubated for 60 min. All incubations were at 37°C.
Activated and nonactivated cells were then used for indirect
fluorescent immunocytochemistry.
Preparation of rabbit anti-mouse USF2 Abs
The preparation of the rabbit anti-mouse USF2 Ab has previously been described (3). Since this anti-USF2 Ab was produced against a 14-amino acid peptide in the C-terminal region of the protein, it is designated in the present paper as anti-USF2-c. The second rabbit anti-mouse USF2 Ab was produced against a 13-amino acid peptide in the middle region of the protein, and, therefore, it is designated as anti-USF2-m. The synthetic 13-amino acid peptide against which the anti-USF2-m was prepared was PEPLAVIQNPFSN. Rabbits were immunized initially i.m., and subsequently s.c., with 1 mg of peptide mixed with RIBI adjuvant (RIBI, ImmunoChem Research). The rabbits received two booster immunizations at 14-day intervals with the same amount of peptide. Seven days after the final booster, the animals were bled and the presence of anti-USF2 Abs was determined.
Serum Abs against the synthetic peptide were assayed by ELISA using 100 ng of peptide in each well and 1 mg of BSA/well as control. The serum was diluted by serial 1:10 dilutions to a dilution of 1:104 and compared with preimmune sera. The end point titer was defined as the serum dilution that gave an OD value of 0.2, this value being 10 times higher than the mean background absorbance (BSA). Detection of Ab was performed using anti-rabbit IgG coupled with alkaline phosphatase, and 1 mg/ml p-nitrophenyl phosphate was used as the substrate for colorimetric detection at 405 nm.
Depletion of Ab reactivity was conducted by preincubating the serum abs at the 1:104 dilution with MC-9 cell extracts before the assay. Prior incubation with MC-9 cell extract led to an 80% reduction in Ab reactivity to the peptide Ag.
Indirect fluorescent immunocytochemistry
Treated cells were cytocentrifuged and fixed with methanol (-20°C) for 3 min. After 45 min blocking with normal goat serum, indirect fluorescent immunocytochemistry was conducted using anti-USF2 or anti-c-Fos (Oncogene Research Products-Calbiochem, La Jolla, CA) Abs and fluorescein (FITC)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). The cells were then counterstained with propidium iodide (PI) and mounted. Specimens were examined, analyzed, and photographed using Sarastro Phoibos (Molecular Dynamics, Sunnyvale, CA) laser scanning confocal microscopy.
The fluorescent intensity of the nuclei was arbitrarily scored by an independent estimation conducted by three observers.
Laser scanning confocal microscopy
Immunofluorescent samples were analyzed using the Sarastro Phoibos 1000 laser scanning confocal microscope (Molecular Dynamics) equipped with a 488-nm argon ion laser and attached to a Universal Zeiss epifluorescence microscope with an oil-immersed Plan Apo 63 x 1.4 N.A. objective lens. FITC labeling was observed with barrier filter 530 DF and PI labeling with barrier filter 570 EFLP. The contrast levels of the images were adjusted by using the Adobe Photoshop program. The images were printed from Codonics NP-1600 Photographic Network Printer at 300 dots per inch (dpi). The possibility of crossover, from one fluorophore channel to the other when collecting both the green (FITC) and the red (PI) confocal images simultaneously in a double channel, was largely eliminated by tuning the gain of each channel.
Peptide synthesis
A 45-amino acid peptide, USF-nuclear translocation inhibitor (USF-NTI), was synthesized, corresponding to the amino acid sequence of a membrane translocating carrier, the signal element of FGF-4 (previously known as K-FGF) AAVALLPAVLLALLAP (9, 10) and that of the USF-specific NLS region, 199–227, MMTPQDVLQTGTQRTIAPRTHPVSPQIDG (5). A search of the SwissProt Data Bank for the USF2-specific NLS peptide homology did not reveal a protein with greater than 10% homology to this peptide. NLS unrelated peptide (NUP), a 41-amino acid peptide corresponding to the same amino acid sequence of a membrane translocating carrier and EILLPNNYNAYESYKYPGMFIALSK, was kindly provided by Dr. J. Hawiger, Nashville, Tennessee.
Peptide labeling and membrane permeability
USF-NTI was labeled with either FITC or with tetramethyl-rhodamine isothiocyanate (TRITC) as described by Goding (11) (TRITC was observed with the same barrier filter as PI). The labeled peptide was added to the cells 30 min before adding IL-3, as described above for cell activation. The cells were mounted immediately after fixation and viewed using the laser scanning confocal microscope. The staining inside the cells came solely from cell membrane penetration of the labeled peptide.
Inhibition of nuclear translocation
Washed cells were incubated in 60-well terasaki trays (Robbins Scientific, Mountain View, CA) for 24 h with or without 1.6 x 10-4 U/ml of IL-3 and with or without 50 µM of either USF-NTI or NUP. Cell viability was determined by trypan blue exclusion.
For the immunocytochemistry, MC-9 and BMMC cells were treated with or without various doses of either USF-NTI or NUP, 30 min before the addition of IL-3.
Measurement of apoptosis
MC-9 cells were washed twice and incubated overnight in growth medium alone, with IL-3, or with IL-3 and USF-NTI (10 µM). Then the cells were labeled with PI, and the ratio of apoptotic cells was determined by FACS (12).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cellular localization of USF2 has been studied in USF2-transfected HeLa cells and was found to be nuclear (5). However, we were interested in the cellular localization of endogenous USF2. Our previous studies have shown that USF2 is highly expressed in mast cells (3). We therefore examined whether activation of murine mast cells, either in a primary culture (BMMC) or in a mast cell line (MC-9), could induce USF2 nuclear translocation.
The cellular localization of the endogenous USF2 was determined by indirect fluorescent immunocytochemistry. The cells were labeled with FITC-conjugated Abs (green) against the highly specific anti-USF2 Abs and counterstained with PI (red) to label their nuclei.
Confirmation of the presence of USF2 in the nucleus was determined by optically scanning the samples at 0.6-mm increases through the z-axis to ensure that the cells were viewed at the mid-nuclear level, thus guaranteeing that the nucleus was not overshadowed by the cytoplasm.
Cells grown in the presence of IL-3 and stained immediately after washing, all had USF2 in their nuclei. When such cells were transferred to RPMI 1640 medium alone and stained after 20 min, most of the USF2 was present in the cytosol rather than in the nucleus (data not shown).
Cells were divided into two groups and incubated for 60 min in RPMI
1640 medium after which time IL-3 was added to one of the groups. When
stained, control cells showed no USF2 in their nuclei. This was shown
by the lack of green staining over the red nuclei in the control (CTRL)
samples in Figures 1
and
2. Exposure of mast cells to IL-3 for 60
min resulted in a definite staining of USF2 inside the nuclei. The
observed yellow staining of the nuclei was caused by an overlap of
green (USF2) staining and red (nuclear) staining (Figs. 1 and 2).
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An investigation into the kinetics of the nuclear translocation of USF2 in response to IL-3 exposure was conducted. No USF2 nuclear translocation was observed after 20 min exposure to IL-3. After 40 min exposure, a fraction of the cells showed light USF2 staining in their nuclei. Most of the cells showed definite USF2 staining in their nuclei only after 60 min incubation with IL-3 (data not shown).
Similar results were obtained when these cells were first sensitized
with IgE and then triggered with DNP-BSA (Ag) (Figs. 1
and 2). However,
IL-3 had no effect on the nuclear translocation of c-Fos in
MC-9 cells (Fig. 3).
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Next it was determined whether this USF2 nuclear transloca-
tion was receptor mediated. The cells were exposed to calcium
ionophore (CI) A23187. This ionophore has been shown to be a potent
mast cell activator that causes degranulation and nuclear signaling
(13). Only a minimal difference was observed in the
subcellular localization of USF2, whether the cells were
activated or not by CI (Fig. 1). Thus, translocation of this
transcription factor from the cytosol into the nucleus occurs only upon
ligand-receptor interaction and not as a result of CI activation.
USF2’s nuclear translocation can be inhibited by a specific peptide
The role played by the USF2-specific NLS in this receptor-mediated
nuclear trafficking was then determined. Recently, peptides containing
signal peptides were successfully used to introduce short peptides into
cells (10, 14, 15, 16). In the present work, a peptide constructed from the
NLS of USF2 (5) and the hydrophobic region of the signal element of
FGF-4 (9, 10), was used as an inhibitor of USF nuclear translocation
(USF-NTI). The FGF-4 signal element has been reported to be effective
as a membrane-translocating carrier, allowing the import of synthetic
peptides into the cell in 3T3 National Institutes of Health fibroblasts
(10). This cross-membrane effect was indeed observed in MC-9 cells that
were treated with USF-NTI labeled with FITC (Fig. 4). Similar results were obtained with
TRITC-labeled USF-NTI (results not shown).
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and 6,
respectively). NUP, which similarly contained the FGF-4 signal element
but whose remaining sequence was unrelated to the USF-specific NLS, was
used as a control for the inhibitory effect of USF-NTI. We have used
this control, which is the original cell-permeable peptide that was
used by Lin’s group to prove the capability of the signal element to
confer cell-membrane permeability, to prove that it is not the mere
insertion of an external peptide that caused the effect, but rather the
NLS sequence that USF-NTI carries. Figure 5 shows that NUP did not
prevent the nuclear translocation of USF2 in BMMCs activated with IL-3.
Thus, USF-NTI specifically blocked the translocation of USF2 into the
nucleus.
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Since we have used fluorescent immunocytochemistry as the sole
approach for following protein nuclear translocation, another
anti-mouse USF2 polyclonal Ab (anti-USF2-m) was prepared and
used to confirm the results obtained with anti-USF2-c. Similar
results were obtained using this anti-USF2-m Ab in BMMC treated
with IL-3 or with IL-3 and USF-NTI (Fig. 7).
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As mentioned above, it is highly likely that USF2 is involved in
the regulation of cell growth. We therefore looked at the effect of
USF-NTI on survival rates in IL-3-activated BMMCs. BMMCs were washed
and preincubated for 1 h in growth medium alone before adding a
suboptimal dose of IL-3, with or without 25 to 100 µM USF-NTI.
Twenty-four hours later, cell viability was determined by trypan blue
exclusion. As shown in Table I,
incubation of these cells with USF-NTI decreased their survival rate in
a dose-dependent fashion by over 70%. No differences in cell death
were observed when the BMMC were treated with a 50-µM concentration
of USF-NTI. Thus, 50 µM of USF-NTI was the concentration that was
chosen for additional experiments in which both BMMC and MC-9 cells
were incubated with 50 µM USF-NTI or 50 µM NUP as a control.
Incubation of MC-9 cells with USF-NTI decreased their survival rate by
over 50% whereas incubation of these cells with the same concentration
of NUP did not have any effect (Table II). Similar results were obtained
with BMMCs (data not shown).
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In complementary experiments, the effect of USF-NTI treatment on the apoptotic response of IL-3-treated MC-9 cells was determined. In USF-NTI-treated cells, a 52 ± 1% (mean ± SE, n = 3) increase in the number of apoptotic cells compared with control was observed (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously observed that the induction of USF2 synthesis
occurred within minutes in cells triggered by mast cell growth factors
or by IgE-Ag. This may indicate that this transcription factor is
involved in the regulation of mast cell growth. Although aggregation of
FcRI in mast cells leads to exocytosis, it also seems to serve as an
effective signal for the regulation of mast cell proliferation rates
(13, 17, 18).
In the present work, we are the first to correlate IL-3-mediated USF2 nuclear translocation with cell viability. Considering the regulatory role of other transcription factors, such as Myc and Max, that have been found to be significantly involved in cell growth (19) and the observation that these transcription factors bind to the same DNA-binding motif as USF2, it is not surprising to find that USF2 is involved in the regulation of cell growth. Furthermore, an indirect indication for the involvement of USF2 in cell division has been recently described (20). It was shown that USF2 may serve as a negative regulator of cell proliferation by antagonizing the transforming function of Myc and also through a more general inhibitory effect on growth.
Since a significant amount of USF2 in the resting cells was found to be attached to the nuclear membrane, the separation of the cytosol from the nuclei and the subsequent determination of the USF2 distribution in each fraction was a complicated maneuver. Thus, to ensure the validity of the use of fluorescent immunocytochemistry as the sole approach for following protein nuclear translocation, we prepared another anti-mouse USF2 polyclonal Ab (anti-USF2-m) and used this Ab to confirm the results obtained with anti-USF2-c. Since similar results were obtained with the newly prepared Ab, it is highly likely that the fluorescent signal observed in the cells using these anti-USF2 Abs was indeed the USF2 protein.
Recently, NLS has been fused to signal peptides that allow the entry of the resultant NLS-competing peptide into the cells (10). Using this approach, it was possible to block the intranuclear translocation of USF2 and its activity as a nuclear factor. However, the USF2 NLS designed shared 100% homology to the NLS site in USF1, and, therefore, this USF-NTI NLS peptide probably also inhibits the nuclear translocation of USF1. Moreover, our observation of USF-NTI-mediated inhibition of mast cell viability could also be explained by the inhibition of USF1 nuclear translocation or the inhibition of the translocation of both USFs.
It is important to note that most of the USF2 protein is found in vivo in a heterodimer form consisting of USF2 and USF1 (21). Only small fractions of USF2 and USF1 are found as homodimers. Thus, it seems logical to assume that the USF2 found in the nuclei of activated mast cells is a heterodimer containing USF1. An intriguing question is whether different forms of such heterodimers of USF2-USF1 could produce different effects on mast cell viability.
The stimuli-dependent nuclear translocation of USF2 observed in mast cells in the present study is in contrast to the findings of Luo and Sawadogo, in which USF2 was found solely in the nucleus of USF2-transfected HeLa cells (5). This can be accounted for not only by the difference in cell type but also by the difference between investigating endogenously expressed genes as opposed to transfected genes.
A greater understanding of the role played by USF2 in mast cell viability may be reached by investigating the possible association of USF2 with other transcription factors. The Zip and the HLH dimerization motifs that are found in various eukaryotic transcription factors characterize these proteins as members of the basic region Zip or bHLH families and permit the formation of homodimers and heterodimers (1, 2, 3, 21). Dimerization of USF2 with members of the bHLH-Zip proteins is theoretically predicted (2) and could be stimulus specific. Therefore, the identification of the profile of USF2-associated proteins from nonactivated, as well as activated, mast cells will undoubtedly provide information regarding the function of USF2.
Our recent observation regarding the ability of USF2 to bind to other nuclear proteins such as c-Fos (3, 22) or mi (23), one of the predominant proteins regulating mast cell growth, might have implications for the diversity of USF2 transcriptional regulation. Moreover, we also observed that a complex containing mi bound to USF2 was detected only in activated mast cells (23). The formation of USF2-Fos or USF2-mi complexes seems to attenuate the activity of USF2 by modulating its affinity to its DNA-binding domain. Alternatively, such a complex, if produced in the cytosol, might decrease the amount of USF2 available in the nucleus. Thus, mi and c-Fos could indirectly control genes whose expression may be regulated by USF2.
The effect of the inhibition of receptor-mediated USF2 nuclear translocation on the process leading to cell death has yet to be determined, as has the involvement of the dimerization of USF2 with other transcription factors in the regulation of mast cell growth. Nevertheless, our findings implicate a role for mast cell surface receptor-mediated USF nuclear translocation in the maintenance of cellular viability.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ehud Razin, Department of Biochemistry, Hebrew University-Hadassah Medical School, P.O.Box 12272, Jerusalem, 91120, Israel. E-mail address:
3 Abbreviations used in this paper: USF2, upstream stimulating factor 2; Zip, leucine zipper; NLS, nuclear localization signal; BMMC, bone marrow-derived mast cells; PI, propidium iodide; NTI, nuclear translocation inhibitor; NUP, NLS unrelated peptide; TRITC, tetramethyl-rhodamine isothiocyanate; CI, calcium ionophore; bHLH, basic region helix-loop-helix.
Received for publication February 3, 1998. Accepted for publication May 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI in mast cells induces the synthesis of Fos-interacting protein and increases its DNA binding activity: the dependence on protein kinase C-ß. J. Biol. Chem. 271:1514.
B by synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J. Biol. Chem. 270:14255.This article has been cited by other articles:
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  K.-Y. Park and A. F. Russo Control of the Calcitonin Gene-related Peptide Enhancer by Upstream Stimulatory Factor in Trigeminal Ganglion Neurons J. Biol. Chem., February 29, 2008; 283(9): 5441 - 5451. [Abstract] [Full Text] [PDF]
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 Y.-N. Lee and E. Razin Nonconventional Involvement of LysRS in the Molecular Mechanism of USF2 Transcriptional Activity in Fc{varepsilon}RI-Activated Mast Cells Mol. Cell. Biol., October 15, 2005; 25(20): 8904 - 8912. [Abstract] [Full Text] [PDF]
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 W. G. Chen, A. E. West, X. Tao, G. Corfas, M. N. Szentirmay, M. Sawadogo, C. Vinson, and M. E. Greenberg Upstream Stimulatory Factors Are Mediators of Ca2+-Responsive Transcription in Neurons J. Neurosci., April 1, 2003; 23(7): 2572 - 2581. [Abstract] [Full Text] [PDF]
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 Y. Fei and T. E. Hughes Nuclear Trafficking of Photoreceptor Protein Crx: The Targeting Sequence and Pathologic Implications Invest. Ophthalmol. Vis. Sci., September 1, 2000; 41(10): 2849 - 2856. [Abstract] [Full Text]
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