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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1540-1548
H-Ras Is Involved in the Inside-out Signaling Pathway of
Interleukin-3-Induced Integrin Activation
By
Hirohiko Shibayama,
Naoyuki Anzai,
Stephen E. Braun,
Seiji Fukuda,
Charlie Mantel, and
Hal E. Broxmeyer
From the Departments of Microbiology/Immunology, Medicine, and the
Walther Oncology Center, Indiana University School of Medicine,
Indianapolis; and the Walther Cancer Institute, Indianapolis, IN.
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ABSTRACT |
The proto-oncogene product, p21ras, has been implicated
in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras,
which is an important component of their growth-promoting signaling
pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)-induced integrin activation in the murine Baf3 cell line after
transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to
fibronectin was induced by IL-3 in a dose-dependent manner via very
late antigen-4 (VLA-4;  4 1 integrins) and VLA-5
(51 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active
H-Ras-transfected Baf3 cells adhered to fibronectin without IL-3
stimulation through VLA-4 and VLA-5, whereas dominant-negative
H-Ras-transfected Baf3 cells showed significantly less adhesion
induced by IL-3 compared with wild-type and constitutively active
H-Ras-transfected Baf3 cells. Anti-1 integrin antibody (clone;
9EG7), which is known to change integrin conformation and activate
integrins, induced the adhesion of dominant-negative
H-Ras-transfected Baf3 cells as much as the other types of
H-Ras-transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1
pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed
proliferation and phosphorylation of MAPK detected by Western blotting
with anti-phospho-MAPK antibody, but not adhesion of any type of
H-Ras-transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC)
inhibitor, suppressed adhesion of these cells completely. These data
indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK
pathway, are involved in the inside-out signaling pathway of
IL-3-induced VLA-4 and VLA-5 activation in Baf3 cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ADHESION OF CELLS TO
extracellular matrix proteins, such as fibronectin (FN), collagen, and
laminin, has profound effects on cell growth, differentiation, and gene
expression. Adhesion of cells to these extracellular matrix proteins is
mediated by integrins. At least 20 different integrins have been
characterized to date, all of which are heterodimeric transmembrane
proteins composed of an  subunit that is noncovalently associated
with a subunit at the cell surface.1 The integrins are
capable of transducing biochemical signals across the plasma membrane to regulate cellular functions; this signaling pathway initiated by
cell-matrix interactions is called outside-in signaling. There are many
reports regarding outside-in signaling, and protein tyrosine phosphorylation has been implicated as playing a central role in this
pathway.2-5
To achieve correct cellular function through cell-matrix interactions,
the interactions between integrins and their ligands need to be
regulated in a number of ways. One way is regulation of expression
levels of integrins at the cell surface. Another is regulation of the
activity of integrins. Integrins are not always able to bind to their
ligands and must be activated by intracellular signals to bind to their
ligands. The cytoplasmic domains of integrins are important for their
activation from inside the cell.6 Stimulating agents such
as phorbol esters, calcium ionophores, chemoattractants, and
aggregation of surface receptors induce activation of integrins. This
signaling pathway is called inside-out signaling.7,8
Recently, some cytokines have been demonstrated to activate integrins.
For example, steel factor induces mast cell adhesion to
FN9; interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and steel factor activate very late
antigen-4 (VLA-4) and VLA-5 on human
hematopoietic cell lines, MO7e and TF-110; and
thrombopoietin activates VLA-4 and VLA-5 on MO7e
cells.11
Protein kinase C (PKC), phospholipase C (PLC), and phosphoinositide
3-OH kinase (PI 3-kinase) are thought to be involved in the inside-out
signaling pathways upstream of the cytoplasmic domains of
integrins.7,8 However, the precise mechanism for these
effects has not been elucidated, and conflicting reports have recently
been published. It has been reported that a constitutively active R-Ras
activates integrins.12 Conversely, it has been reported
that a constitutively active H-Ras inactivates integrins.13 Because Ras is activated via many cytokines, there is a possibility that it may have some role in inside-out signaling induced by these
cytokines. No information about Ras involvement in cytokine induced
inside-out signaling pathway has thus far been reported. For this
reason, we investigated the function of H-Ras in the inside-out
signaling pathway of cytokine-induced integrin activation. In a
previous study,14 we found that IL-3 activated VLA-4 and VLA-5 on the murine hematopoietic cell line, Baf3, and induced adhesion
of these cells to FN. In this study, we used constitutively active,
dominant-negative, and wild-type H-Ras-transfected Baf3 cells to
examine the effects of H-Ras on the adhesion of these cells to FN.
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MATERIALS AND METHODS |
Antibodies, cytokines, chemicals, and plasmids.
Anti-1 integrin antibodies (Ha2/5 and 9EG7), anti-4 integrin
(R1-2), anti-5 integrin (5H10-27), anti-v integrin (H9.2B8), their isotype-matched control antibodies, fluorescein isothiocyanate (FITC)-conjugated goat antirat IgG, and FITC-conjugated
mouse antihamster IgG were purchased from Pharmingen (San Diego, CA). Anti-H-Ras antibody (F235) was purchased from Santa Cruz
Biotechnology, Inc (Santa Cruz, CA). Anti-glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) antibody (6C5) was purchased from Biodesign Int
(Kennebunk, ME). Anti-phospho-p44/42 MAPK antibody and anti-p44/42
MAPK antibody were purchased from New England Biolabs, Inc (Beverly,
MA). Recombinant mouse IL-3 and mouse IL-4 were purchased from R&D
Systems, Inc (Minneapolis, MN). Human fibronectin was purchased from
Collaborative Biochemical (Bedford, MA). 8-bromo adenosine
3':5'-cyclic monophosphate (8-Br-cAMP), N6,
2'-O-dibutyryl adenosine 3':5'-cyclic monophosphate
(Dibutyryl-cAMP), and wortmannin were purchased from Sigma Chemical Co
(St Louis, MO). H-7, U-73122, and U-73343 were purchased from
Calbiochem (San Diego, CA). PD98059 was purchased from New England
BioLabs Inc. pUSE H-Ras (a constitutively active type, a
dominant-negative type, and the wild-type), eukaryotic expression
vector containing H-Ras (a constitutively active type, a
dominant-negative type, and the wild-type) cDNA under the control of
the cytomegalovirus (CMV) promotor, and the neomycin
resistance gene with the SV40 promotor were purchased from Upstate
Biotechnology (Lake Placid, NY). The constitutively active type H-Ras
mutation is a substitution of leucine for glutamine at position
61.15 The dominant-negative type H-Ras mutation is a
substitution of asparagine for serine at position 17.16
Cells and cell culture.
The murine hematopoietic cell line, Baf3,17 a
factor-dependent cell line requiring IL-3 or IL-4 for both growth and
survival was used. Baf3 cells were cultured in RPMI-1640 medium
supplemented with 10% fetal calf serum (FCS) and 100 pg/mL mouse IL-3
or 1 ng/mL mouse IL-4. The optimal concentrations of these cytokines were determined in preliminary experiments. The plasmids were transfected into Baf3 cells by electroporation. Stable transfectants were selected by antibiotic G418 (1 mg/mL; GIBCO BRL, Grand Island, NY)
and analyzed. These cells were washed and incubated in RPMI-1640 medium
with 1% bovine serum albumin (BSA; Sigma) without growth factors for
16 to 18 hours to growth factor-starve the cells and were used for the
following experiments.
[3H]-thymidine incorporation assay.
The growth factor-starved Baf3 and all transfected Baf3 cells in
RPMI-1640 medium with 10% FCS were plated (0.2 mL/well) at a density
of 2 × 104 cells/well in 96-well tissue culture
flat-bottom plates (Corning-Costar, Ann Arbor, MI) with or without IL-3
or IL-4 and cultured for 2 days. DNA synthesis was determined by the
addition of [3H]-thymidine (0.5 µCi/well; Amersham,
Arlington Heights, IL) for the final 6 hours of the culture. The cells
were harvested onto glass-fiber filters and the amount of incorporated
radioactivity was determined by liquid scintillation counting.
Adhesion assay.
Human FN and BSA were diluted in phosphate-buffered saline (PBS). One
hundred microliters of 20 µg/mL FN was distributed in 96-well tissue
culture flat-bottom plates. After overnight incubation at 4°C, the
coated wells were washed twice with PBS and 100 µL of PBS with 1%
BSA was added and plates were incubated at 37°C for 1 hour to block
nonspecific binding. At the same time, 100 µL of PBS with 1% BSA was
distributed in other wells and incubated at 37°C for 1 hour to act
as control wells. The wells were then washed twice with PBS. The growth
factor-starved Baf3 and all transfected Baf3 cells were used for the
adhesion assay. The cells were labeled with 51Cr (100 µCi/3 × 107 cells; Amersham) for 1 hour at 37°C
with RPMI-1640 medium containing 1% BSA. Cells were washed twice and
resuspended at 3 × 106 cells/mL in the same
conditions. A total of 100 µL of the cell suspension was added to
each of the FN-coated wells and BSA-coated wells, which were
centrifuged at 600 rpm for 1 minute to allow attachment of cells to the
bottom of the wells, followed by incubation for 30 minutes at 37°C.
Unattached cells were removed by washing twice with prewarmed RPMI-1640
medium containing 1% BSA. Adherent cells were solubilized with 1%
sodium dodecyl sulfate, and radioactivity was quantitated in a  counter. The percentage of adherent cells (percentage of input) was
determined by dividing the activity in the adherent fraction by the
radioactivity contained in 100 µL of the initial labeled cell
suspension as previously described.18
Cell lysis and immunoblotting.
Cells were lysed in lysis buffer (20 mmol/L Tris-HCl, pH 8.0, 137 mmol/L NaCl, 10% glycerol, 1% NP-40, 1 mmol/L phenylmethylsulfonyl fluoride, 0.15 U/mL aprotinin, 10 mmol/L EDTA, 10 µg/mL leupeptin, 100 mmol/L sodium fluoride, and 2 mmol/L sodium orthovanadate). After
30 minutes on ice, insoluble fractions were removed by centrifugation at 14,000 rpm for 10 minutes (whole cell lysate). Whole cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride
(PVDF) membrane (Millipore Corp, Bedford, MA). Membranes were blocked in Tris-buffered saline containing 0.5% Tween 20 and 2% BSA at room
temperature for 1.5 hours and incubated with appropriate primary
antibodies for 1.5 hours. Blots were visualized using horseradish
peroxidase (HRP)-conjugated secondary antibodies and an
enhanced chemiluminescent system (ECL; Amersham). To reprobe with
another primary antibody, membranes were incubated in stripping buffer
(62.5 mmol/L Tris, pH 6.7, 100 mmol/L 2-mercaptoethanol, 2% SDS) at
70°C for 1 hour, washed, and then used for further study.
Flow cytometric analysis.
Cells (5 × 105) were incubated with 2 µg of
antibody at 4°C for 30 minutes and then washed twice in PBS. Cells
were incubated with 0.4 µL of FITC-conjugated goat antirat IgG or
FITC-conjugated mouse antihamster IgG at 4°C for 30 minutes, washed
twice in PBS, and analyzed by flow cytometry using a FACScan flow
cytometer (Becton Dickinson, Sunnyvale, CA).
Inhibition of adhesion by anti-integrin antibodies.
Using anti-integrin antibodies (Abs; anti-1 integrin [clone:
Ha2/5], anti-4 integrin, anti-5 integrin, and anti-v
integrin), we evaluated the role of each FN receptor for cell adhesion
by the ability of each antibody to inhibit adhesion. A concentration of
10 µg/mL of each antibody, which was found to be an effective amount,
was used for the inhibition assays. The growth factor-starved transfected Baf3 cells were labeled with 51Cr and incubated
at 37°C for 30 minutes in the absence or presence of these Abs or
their isotype-matched control Abs. Cells were subsequently transferred
to FN-coated wells and incubated for an additional 30 minutes at
37°C with or without 0.1 ng/mL IL-3, and the percentage of adherent
cells was measured as noted above.
Inhibition of proliferation and adhesion by the inhibitors of Ras,
Raf-1, MAPK kinase, and MAPK pathway.
To investigate downstream events of H-Ras in the inside-out signaling
pathway of IL-3-induced integrin activation, the effects of inhibitory
compounds of Ras, Raf-1, MAPK kinase, and MAPK pathway were evaulated.
We selected 8-Br-cAMP and Dibutyryl-cAMP as inhibitors of the Ras-Raf-1
pathway19,20 and PD98059 as an inhibitor of MAPK
kinase.21 To examine the effects of these inhibitors on proliferation, the growth factor-starved transfected Baf3 cells were
precultured with or without 1 mmol/L 8-Br-cAMP or 1 mmol/L Dibutyryl-cAMP or 10 and 50 µmol/L PD98059 for 1 hour, and the cells
were used for [3H]-thymidine incorporation assay as
described above. To examine the effects of these inhibitors on
adhesion, the growth factor-starved transfected Baf3 cells were labeled
with 51Cr and incubated at 37°C for 30 minutes to 3 hours in the absence or presence of 1 mmol/L 8-Br-cAMP or 1 mmol/L
Dibutyryl-cAMP, or 10 and 50 µmol/L PD98059. Cells were subsequently
transferred to FN-coated wells and incubated for an additional 30 minutes at 37°C with or without 0.1 ng/mL IL-3, and the percentage
of adherent cells was measured as noted above. The optimal
concentrations of each inhibitor compound tested were determined in
preliminary experiments.
Inhibition of adhesion by inhibitor compounds.
To examine the potential mechanisms of inside-out signaling of
IL-3-induced cell adhesion to FN, the effects of different inhibitor
drugs on adhesion were tested. PI-3 kinase inhibitor, wortmannin (100 nmol/L), PKC inhibitor, H-7 (50 µmol/L), and PLC inhibitor,
U-7312222 (1 µmol/L), were used as inhibitor compounds. U-73433 (1 µmol/L), a close analog of U-73122, was also tested. The
growth factor-starved transfected Baf3 cells were labeled with
51Cr and incubated at 37°C for 30 minutes in the
absence or presence of those inhibitor compounds. Cells were
subsequently transferred to FN-coated wells and incubated for an
additional 30 minutes at 37°C with or without 0.1 ng/mL IL-3, and
the percentage of adherent cells was measured as noted above. The
optimal concentrations of each inhibitor compound tested were
determined in preliminary experiments.
Adhesion by anti-1 integrin antibody (clone; 9EG7).
Anti-1 integrin antibody (clone; 9EG7) is known to change the
integrin conformation from outside of cells and induce the cells'
adhesion.23 Using this antibody, we evaluated effects of
H-Ras on cell cytoskeleton or signal transduction pathways from the
integrins that need to be activated for cells to adhere to FN. A
concentration of 5 µg/mL of this antibody, which was found to be an
effective amount, was used for the adhesion assay. The growth
factor-starved Baf3 and all transfected Baf3 cells were labeled with
51Cr and incubated at 37°C for 30 minutes in the
presence of this antibody or isotyped-matched control antibody. Cells
were subsequently transferred to FN-coated wells and incubated at
37°C for an additional 30 minutes, and the percentage of adherent
cells was measured as noted above.
Statistical analysis.
Statistical analysis was performed using a two-sample t-test.
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RESULTS |
H-Ras expression.
As shown in Fig 1, overexpression of H-Ras
was seen in all types of H-Ras-transfected Baf3 cells compared with
nontransfected Baf3 cells.
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| Fig 1.
H-Ras expression in nontransfected Baf3 cells (designated
as Baf3/c) and wild-type, constitutively active type, and
dominant-negative type H-Ras-transfected Baf3 cells (designated as
wild-type, constitutively active, and dominant-negative, respectively).
Cells were lysed in lysis buffer, separated by 15% SDS-PAGE,
immunoblotted with anti-H-Ras antibody, and then stripped and
reblotted with anti-GAPDH antibody (as an internal control). This is a
representative result of three independent experiments.
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Proliferation capacity.
As shown in Fig 2A, the wild-type
H-Ras-transfected Baf3 cells proliferated in the same IL-3
dose-dependent manner as nontransfected Baf3 cells. The constitutively
active type H-Ras-transfected Baf3 cells proliferated without IL-3
stimulation. IL-3 induced additional proliferation of the
constitutively active type H-Ras-transfected Baf3 cells. In high
concentrations of IL-3 (>0.1 ng/mL), the proliferation capacity of
the constitutively active type H-Ras-transfected Baf3 cells was the
same as the other types of Baf3 cells. The proliferation capacity of
the dominant-negative type H-Ras-transfected Baf3 cells was the same
as the wild-type H-Ras-transfected Baf3 cells and the nontransfected
Baf3 cells. These data are consistent with the data of others showing
that this same dominant-negative type H-Ras exhibited no inhibitory
effect on IL-3-dependent proliferation of Baf3 cells, as assessed by
an increase in cell numbers and mitochondrial enzyme
activity.24 We also investigated the proliferation effects
of IL-4 in these cell lines. As shown in
Fig 3A, the nontransfected Baf3 cells and
all types of H-Ras-transfected Baf3 cells had a proliferative response
to IL-4 stimulation. This result serves as control for studies reported
in the next section.
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| Fig 2.
[3H]-thymidine incorporation (A) and
adhesion to FN (B) of nontransfected Baf3 cells (designated as Baf3/c),
wild-type H-Ras-transfected Baf3 cells (designated as wild-type),
constitutively active type H-Ras-transfected Baf3 cells (designated as
constitutively active), and dominant-negative type H-Ras-transfected
Baf3 cells (designated as dominant-negative) in the presence or absence
of various concentrations of IL-3. Data represent the mean (±SD) of
triplicate samples from one of three representative experiments.
*P value (P < .01) comparing proliferation or
adhesion with those of wild-type H-Ras-transfected Baf3 cells.
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| Fig 3.
[3H]-thymidine incorporation (A) and
adhesion to FN (B) of nontransfected Baf3 cells (designated as Baf3/c),
wild-type H-Ras-transfected Baf3 cells (designated as wild-type),
constitutively active type H-Ras-transfected Baf3 cells (designated as
constitutively active), and dominant-negative type H-Ras-transfected
Baf3 cells (designated as dominant-negative) in the presence or absence
of IL-4 or IL-3 (as a control). Data represent the mean (±SD) of
triplicate samples from one of three representative experiments.
*P value (P < .01) comparing proliferation or
adhesion induced by cytokines with those of no cytokine stimulation.
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Adhesion capacity.
As shown in Fig 2B, the wild-type H-Ras-transfected Baf3 cells adhered
to FN in the same manner as nontransfected Baf3 cells in low
concentrations of IL-3 (<0.01 ng/mL), whereas in high concentrations of IL-3 (>0.1 ng/mL), the adhesion of the wild-type
H-Ras-transfected Baf3 cells increased significantly compared with
nontransfected Baf3 cells. The constitutively active type
H-Ras-transfected Baf3 cells adhered to FN without IL-3 stimulation.
IL-3 induced additional adhesion to FN of the constitutively active
type H-Ras-transfected Baf3 cells, and in high concentrations of IL-3
(>0.01 ng/mL), the adhesion capacity of the constitutively active
type H-Ras-transfected Baf3 cells was same as the wild-type
H-Ras-transfected Baf3 cells. The adhesion capacity of the
dominant-negative type H-Ras-transfected Baf3 cells was significantly
less than that of the other types of Baf3 cells. As shown in Fig 3B,
neither nontransfected Baf3 cells nor any type of H-Ras-transfected
Baf3 cells showed any significant adhesion to FN in response to IL-4
stimulation. However, Baf3 cells, which proliferate in medium
containing IL-4 (see Fig 3A), did show adhesion to FN induced by IL-3
in the same manner as the Baf3 cells maintained in medium containing
IL-3.
Integrin expression and function.
Expression of integrins (1, 4, 5, and v) was analyzed by
flow cytometry. Baf3 cells expressed integrin 1, 4, and 5, whereas there was no expression of v integrin on the cells. As shown
in Fig 4A, there were no remarkable
differences in expression of any of these integrins between
nontransfected Baf3 cells and any type of H-Ras-transfected Baf3
cells. We have previously shown using blocking Abs against
integrin-1, 4, 5, and v that IL-3 activated VLA-4 and VLA-5
on the Baf3 cells and induced the adhesion of these cells to
FN.14 To analyze the role of integrins in adhesion of
transfected Baf3 cells to FN, inhibition of transfected Baf3 cells
adhesion to FN was analyzed using blocking Abs against integrin-1,
4, 5, and v. As shown in Fig 4B, anti-1 integrin antibody
blocked the constitutively active type H-Ras-transfected Baf3 cells
adhesion to FN and also completely blocked any type of
H-Ras-transfected Baf3 cells adhesion to FN induced by IL-3. Whereas
anti-4 integrin antibody or anti-5 integrin antibody blocked
adhesion only partially, simultaneous addition of both anti-4
integrin and anti-5 integrin antibodies blocked adhesion completely.
Anti-v integrin antibody showed no inhibitory effect on adhesion.
These results suggest that adhesion of any type of H-Ras-transfected
Baf3 cells is mediated by activation of VLA-4 (41) and VLA-5
(51) integrins.
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| Fig 4.
Integrin expression and function. In (A), expression of
integrins (1, 4, 5, and v) was analyzed by flow cytometry.
Nontransfected Baf3 cells (designated as Baf3/c), wild-type
H-Ras-transfected Baf3 cells (designated as wild-type), constitutively
active type H-Ras-transfected Baf3 cells (designated as constitutively
active), and dominant-negative type H-Ras-transfected Baf3 cells
(designated as dominant-negative) expressed integrin 1, 4, and
5, whereas there was no expression of v integrin on the cells.
There were no remarkable differences in any of these integrins between
nontransfected Baf3 cells and any type of H-Ras-transfected Baf3
cells. In (B), inhibition of constitutively active type
H-Ras-transfected Baf3 cells adhesion to FN without IL-3 stimulation
and constitutively active type H-Ras-transfected Baf3 cells, wild-type
H-Ras-transfected Baf3 cells, and dominant-negative type
H-Ras-transfected Baf3 cells adhesion to FN induced by IL-3 by
blocking Abs against integrin-4, 5, and 1 was examined. Data
represent the means (±SD) of triplicate samples from a representative
experiment of three. None of the Abs had an effect on viability of the
cells. *P value (P < .01) comparing adhesion blocked
by each anti-integrin antibody with that of a control antibody.
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8-Br-cAMP, Dibutyryl-cAMP (cAMP), and PD98059 inhibit proliferation
and phosphorylation of MAPK but not adhesion of H-Ras-transfected Baf3
cells.
As shown in Fig 5A, both cAMP and PD98059
inhibited proliferation of the constitutively active type
H-Ras-transfected Baf3 cells. We also examined effects of these
compounds on phosphorylation of MAPK by Western blotting with
anti-phospho-MAPK antibody, which only recognizes phosphorylated MAPK.
As shown in Fig 5B, cAMP and PD98059 suppressed phosphorylation of MAPK
of the constitutively active type H-Ras-transfected Baf3 cells and all
types of H-Ras-transfected Baf3 cells stimulated by IL-3. These
results strongly suggest that cAMP and PD98059 had inhibitory effects
on Ras, Raf-1, MAPK kinase, and the MAPK pathway. On the other hand,
interestingly, cAMP, but not PD98059, inhibited IL-3-induced
proliferation of any type of H-Ras-transfected Baf3 cells. However,
cAMP and PD98059 showed no inhibitory effects on adhesion to FN of any
type of H-Ras-transfected Baf3 cells (Fig 5C). These results indicate that Raf-1, MAPK kinase, and the MAPK pathway might not be involved in
inside-out signaling of IL-3-induced integrins' activation in Baf3
cells.
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| Fig 5.
Effects of Raf-1, MAPK kinase, and MAPK pathway inhibitor
compounds on the proliferation (A), the phosphorylation of MAPK (B),
and the adhesion (C) of constitutively active type H-Ras-transfected
Baf3 cells (designated as constitutively active) without IL-3
stimulation and constitutively active type H-Ras-transfected Baf3
cells, wild-type H-Ras-transfected Baf3 cells (designated as
wild-type), and dominant-negative type H-Ras-transfected Baf3 cells
(designated as dominant-negative) induced by IL-3. In (A),
[3H]-thymidine incorporation of those transfected Baf3
cells in the absence or presence of either 1 mmol/L 8-Br-cAMP, 1 mmol/L
Dibutyryl-cAMP, or 50 µmol/L PD98059 was measured. Data represent the
means (±SD) of triplicate samples from a representative experiment of
three. In (B), the factor-starved transfected Baf3 cells were incubated
at 37°C for 3 hours in the absence or presence of either 1 mmol/L
8-Br-cAMP, 1 mmol/L Dibutyryl-cAMP, or 50 µmol/L PD98059 and
incubated for an additional 15 minutes with or without 0.1 ng/mL IL-3.
Cells were lysed in lysis buffer, separated by 11.25% SDS-PAGE,
immunoblotted with anti-phospho-MAPK antibody, and reblotted with
anti-MAPK antibody to detect the total MAPK levels. Anti-MAPK antibody
used here recognized only p42 MAPK of Baf3 cells. This is a
representative result of three independent experiments. In (C),
transfected Baf3 cells were labeled with 51Cr and incubated
at 37°C for 3 hours in the absence or presence of either 1 mmol/L
8-Br-cAMP, 1 mmol/L Dibutyryl-cAMP, or 50 µmol/L PD98059. Cells were
subsequently transferred to FN-coated wells and incubated at 37°C
for an additional 30 minutes with or without 0.1 ng/mL IL-3, and the
percentage of adherent cells was measured. Data represent the means
(±SD) of triplicate samples from a representative experiment of
three. *P value (P < .01) comparing each treatment
with the control.
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U-73122 (a PLC inhibitor) suppresses adhesion of transfected cells to
FN.
In our previous study,14 neither PI-3 kinase inhibitor,
wortmannin, nor PKC inhibitor, H-7, inhibited IL-3-induced adhesion of
Baf3 cells to FN. However, PLC inhibitor, U-73122, did suppress this
adhesion. The effects of these same inhibitory compounds were tested on
the adhesion of the transfected Baf3 cells. As shown in
Fig 6, wortmannin and H-7 did not suppress
constitutively active type H-Ras-transfected Baf3 cells' adhesion or
adhesion of any type of H-Ras-transfected Baf3 cells induced by IL-3.
The wortmannin used here showed inhibitory activity on another
cytokine-induced cell adhesion system. In contrast, U-73122 did
suppress this adhesion. U-73433, a closely related analog of U-73122
that does not inhibit PLC, showed no inhibitory effect on adhesion.
This suggests that PLC, but not PI3-kinase or PKC, is involved in
inside-out signaling of IL-3-induced integrin activation in Baf3
cells. We examined the effects of U-73122 on IL-3-induced cell
survival using merocyanine 540 (MC540) staining, which can detect the
early apoptotic state of cells,25 and found that there was
no difference in the percentage of apoptotic cells with and without
U-73122.
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| Fig 6.
Effects of inhibitor compounds on constitutively active
type H-Ras-transfected Baf3 cells (designated as constitutively
active) adhesion to FN without IL-3 stimulation and constitutively
active type H-Ras-transfected Baf3 cells, wild-type H-Ras-transfected
Baf3 cells (designated as wild-type), and dominant-negative type
H-Ras-transfected Baf3 cells (designated as dominant-negative)
adhesion to FN induced by IL-3. Those transfected Baf3 cells were
labeled with 51Cr and incubated at 37°C for 30 minutes
in the absence or presence of 100 nmol/L wortmannin, 1 µmol/L
U-73122, 1 µmol/L U-73433, or 50 µmol/L H-7. Cells were
subsequently transferred to FN-coated wells and incubated for an
additional 30 minutes at 37°C with or without 0.1 ng/mL IL-3, and
the percentage of adherent cells was measured. Data represent the means
(±SD) of triplicate samples from a representative experiment of
three. None of the inhibitor compounds had an effect on viability of
the cells. *P value (P < .01) comparing adhesion
blocked by U-73122 with control adhesion.
|
|
Anti-1 integrin antibody (clone; 9EG7) induces
adhesion of any type of Baf3 cells to FN.
As shown in Fig 7, the anti-1 integrin
antibody induced the adhesion to FN of the dominant-negative type
H-Ras-transfected Baf3 cells as efficiently as nontransfected Baf3
cells as well as the other types of H-Ras-transfected Baf3 cells.
These data suggest that H-Ras may not affect cell cytoskeleton or the
signal transduction pathways from the integrins that need to be
activated for these cells to adhere to FN. However, this does support a role of H-Ras in inside-out signals.
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| Fig 7.
Induction of the adhesion to FN of nontransfected Baf3
cells (designated as Baf3/c), wild-type H-Ras-transfected Baf3 cells
(designated as wild-type), constitutively active type
H-Ras-transfected Baf3 cells (designated as constitutively active),
and dominant-negative type H-Ras-transfected Baf3 cells (designated as
dominant-negative) by anti-1 integrin antibody (clone; 9EG7). These
Baf3 cells were labeled with 51Cr and incubated at 37°C
for 30 minutes in the presence of 5 µg/mL of anti-1 integrin
antibody (clone; 9EG7) or isotype-matched control antibody. Cells were
subsequently transferred to FN-coated wells and incubated at 37°C
for an additional 30 minutes, and the percentage of adherent cells was
measured. Data represent the means (±SD) of triplicate samples from a
representative experiment of three. *P value (P < .01) comparing adhesion induced by anti-1 integrin antibody with the
control antibody.
|
|
| |
DISCUSSION |
In the present study, we investigated the function of H-Ras in
the cytokine-induced integrin activation. IL-3, but not IL-4, induced
the adhesion of Baf3 cells to FN. H-Ras is known to be activated by
IL-3, but not IL-4.26,27 We also examined H-Ras activity
with IL-3 stimulation using a Ras-GTP binding assay and found that
H-Ras was activated by IL-3 in Baf3 cells (data not shown).
Furthermore, the dominant-negative type H-Ras-transfected Baf3 cells
showed significantly less adhesion induced by IL-3 compared with the
wild-type and the constitutively active type H-Ras-transfected Baf3
cells. On the other hand, anti-1 integrin antibody (clone; 9EG7),
which is known to change integrin conformation from outside of the
cells and activate, induced the adhesion of the dominant-negative type
H-Ras-transfected Baf3 cells as much as the other types of
H-Ras-transfected Baf3 cells. From these data, it is demonstrated that
H-Ras is involved in the inside-out signaling pathway of IL-3-induced
integrin activation in Baf3 cells.
To examine downstream events of H-Ras in the inside-out signaling
pathway of IL-3-induced integrin activation, we focused on the roles
of Ras, Raf-1, MAPK kinase, and the MAPK pathway. We used 8-Br-cAMP and
Dibutyryl-cAMP as inhibitors of the Ras-Raf-1 pathway and PD98059 as an
MAPK kinase inhibitor and tested the effects of these inhibitors on the
adhesion of the transfected Baf3 cell lines. These inhibitors showed no
suppressive effects on adhesion, which suggests that Ras, Raf-1, MAPK
kinase, and the MAPK pathway might not be involved in the downstream
events of H-Ras in the inside-out signaling pathway of IL-3-induced
integrin activation in Baf3 cells. Our data suggest that H-Ras, but not Ras, Raf-1, MAPK kinase, and the MAPK pathway, is involved in the
inside-out signaling pathway of IL-3-induced integrin activation in
Baf3 cells.
On the other hand, the constitutively active type H-Ras-transfected
Baf3 cell line was able to adhere to FN without IL-3 stimulation via
activation of VLA-4 and VLA-5. This suggests that H-Ras can activate
integrins of Baf3 cells. Recently, it has been reported that H-Ras
suppresses integrin activation,13 data opposite from our
results. One possibility for this conflict in results in the function
of H-Ras might reflect differences in the cells tested. The other
investigators13 used CHO cells transfected with integrin cDNAs, which expressed active forms of integrins. Our study used the
hematopoietic cell line Baf3, which expresses inactive forms of
integrins without IL-3 stimulation. Another possibility is the
difference of targeted integrins of H-Ras. The other investigators examined the effect of H-Ras on IIb3
integrins, whereas we focused on the effect of H-Ras on 41
(VLA-4) and 51 (VLA-5) integrins. Still other investigators
reported that R-Ras activates integrins.12 In that report,
they showed that constitutively active type H-Ras, as a control study,
could not activate integrins. The difference between their
data12 and ours might also depend on difference in the
types of cells tested. Another difference might be the mutation of
H-Ras used. Both groups used constitutively active type H-Ras, which is
a mutation in which a substitution of valine for glycine is made at
position 12. We used the constitutively active type H-Ras, in which the
mutation is a substitution of leucine for glutamine at position 61. H-Ras and R-Ras are highly homologous to one another,28 and
they are known to have some of the same functions, such as
antiapoptotic effects induced by cytokine deprivation.29
Our data demonstrated that H-Ras has another function similar to that
of R-Ras, which is the activation of integrins.
PI-3 kinase is thought to be involved in the inside-out signaling
pathway.30,31 This was demonstrated by the inhibition of
integrin activation by a PI-3 kinase inhibitor,
wortmannin.32 In other integrin activation models,
wortmannin was shown to inhibit adhesion. In our previous study,
wortmannin showed no inhibitory effect on Baf3 cell adhesion to FN
induced by IL-3.14 From these data, PI-3 kinase might not
be involved in this inside-out signaling model of IL-3-induced
integrin activation in Baf3 cells. However, because there is a report
that PI-3 kinase is downstream of Ras,33 we examined the
effect of wortmannin on the adhesion of the constitutively active type
H-Ras-transfected Baf3 cells and found that wortmannin showed no
inhibitory effect. Wortmannin also showed no inhibitory effects on the
adhesion of the wild-type and the dominant-negative type
H-Ras-transfected Baf3 cell lines induced by IL-3. Based on these
data, we believe that PI-3 kinase is not involved in the inside-out
signaling of IL-3-induced integrin activation in the Baf3 cell line we
are using. Because our findings about PI-3 kinase, both previous and
present, are very clear, it is difficult to reconcile this
contradiction to some previous reports at this time. Further study of
this discrepancy within the context of other model systems could be revealing.
On the other hand, PLC is also thought to be involved in the inside-out
signaling pathway.11,34 In our previous study, a PLC
inhibitor, U-73122, completely inhibited Baf3 cell adhesion to FN
induced by IL-3. Thus, we decided to examine the effect of U-73122 on
the adhesion of transfected Baf3 cells. We found that U-73122
suppressed the constitutively active type H-Ras-transfected Baf3 cell
adhesion and adhesion of wild-type and dominant-negative type
H-Ras-transfected Baf3 cells induced by IL-3. The result that U-73122
inhibited the adhesion of any type of H-Ras-transfected Baf3 cells
suggests that PLC might be downstream of H-Ras in the inside-out
signaling pathway of IL-3-induced integrin activation in Baf3 cells.
Ras has been reported to be involved in outside-in signaling pathways
initiated by cell-matrix interactions.4,35 Taking this and
our own data into consideration, Ras appears to be involved in both
inside-out signaling and outside-in signaling pathways of cell-matrix
interactions and might play a central role in cellular functions
regulated by cell-matrix interactions.
| |
FOOTNOTES |
Submitted June 8, 1998; accepted October 20, 1998.
Supported by US Public Health Service Grants No. R01 DK53674, R01
HL56416, and R01 HL54037; by a project in P01 HL53586 from the National
Institutes of Health to H.E.B.; and by the scholarship for young
investigator travel grant for 1997 Osaka University Medical School Fund
for International Exchange.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Hal E. Broxmeyer, PhD, Walther Oncology
Center, Indiana University School of Medicine, 1044 W Walnut St, Room
302, Indianapolis, IN 46202; e-mail: hbroxmey{at}iupui.edu.
| |
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Top
Abstract
Introduction