 |
INTRODUCTION |
Melanoma growth stimulatory activity/growth-regulated protein
(MGSA/GRO)1 is a member of
the CXC chemokine (chemokine with the first two conserved cysteine
residues separated by an intervening amino acid) family.
MGSA/GRO plays a fundamental role in recruitment and activation of
neutrophils, lymphocytes, and monocytes in host defense (1). There are
four genes for MGSA/GRO. Three of them encode closely related proteins
(MGSA/GRO
,
, and
) (2-5), and the other is a pseudogene
(MGSA/GRO
) (6). All three MGSA/GRO proteins bind to the CXC
chemokine receptor designated CXCR2, which is a seven-transmembrane G
protein-coupled receptor. The order of potency for these three
chemokines regarding neutrophil and basophil chemotactic activity,
Ca+2 flux, respiratory burst, exocytosis, shape change, and
receptor binding is MGSA/GRO
>
>
(7).
MGSA/GRO
is expressed in 70% of the human melanoma tumors, but
there is little expression of MGSA/GRO
or
(8). It has become
apparent that MGSA/GRO plays an important role in tumorigenesis and
angiogenesis (8, 9). Aberrant overexpression of MGSA/GRO
has been
implicated in melanoma tumor progression both in vitro and
in vivo (10-13). Mouse immortalized melanocytes (parental
melan-a) stably transfected with MGSA/GRO
,
, or
exhibit an
enhanced ability to form large colonies in soft agar and form melanoma tumors in nude mice (10, 11), as compared with parental melan-a cells
that do not form tumors in nude mice or in C57B1/6 syngenic mice
(14).
Activation of the phosphatidylinositol 3-kinase/Ras/Raf/Soc/MEK1/ERK
pathway is common for G protein-coupled receptors (15-17). Receptors
for chemokines are traditionally considered to be responsible for the
activation of special leukocyte functions such as chemotaxis, degranulation, and the release of superoxide anions. For example, SDF-1
induces tyrosine phosphorylation and association of components of focal adhesion complexes and activates phosphatidylinositol 3-kinase, ERK, and NF-
B in a model of a murine pre-B cell line transfected with the human CXCR4 receptor (18). Interleukin-8 activation of the phosphatidylinositol 3-kinase/Ras/Raf pathway is
required for human neutrophil migration. However, the regulation of
cell migration by interleukin-8 is independent of ERK activation (19).
For some cell types, MGSA/GRO activation of ERK can also be through a
Ras/Raf1-independent pathway (20). However, the underlying mechanisms
for signal transduction from CXCR2 receptors to transcription factors
are poorly understood.
MAP kinase pathways are cellular signaling pathways that enable cells
to transduce extracellular signals to an intracellular response. To
date, four separate MAP kinases have been identified in mammalian
cells: ERK, JNK, p38, and BMK1/ERK5. MAP kinases are activated by
phosphorylation of Thr and Tyr amino acids by dual specificity MAP
kinase kinases (MEKs), which are themselves activated by MAP kinase
kinase kinases (MEKKs). MEKKs are the protein serine-threonine kinases
that have regulatory sequences for binding the small guanine
nucleotide-binding proteins Ras (21) and Cdc42/Rac (22). It has been
reported that MEKK1 is one of several downstream effectors of Ras (21).
Except for MEK4, MEKs are, in general, very specific for downstream MAP
kinases. MEK1 and MEK2 selectively phosphorylate and activate the ERK
subgroup (23), whereas MEK3 and MEK6 selectively activate p38 (24-26). MEK4 does not activate the ERK subgroup but activates both p38 and JNK
(24, 27), whereas MEK5 activates BMK1/ERK5 (28). MEK7 has been
identified as a specific activator of the JNK subgroup (29, 30). In
contrast, the activities of MEKKs show less specificity, and each of
the MEKKs transduce signals to more than one MAP kinase cascade.
Activation of Ras also has been implicated in the control of NF-
B
activities in fibroblasts (31). This NF-
B activation is required for
Ras-initiated cellular transformation in NIH 3T3 cells (31, 32).
Inhibition of NF-
B by I
B
expression blocks focus formation
induced by oncogenic Ras in NIH 3T3 cells (31). In addition, the
expression of antisense p65 blocks cellular transformation (33, 34). It
has been reported that there is enhanced NF-
B transcriptional
activity through Raf-dependent and Raf-independent mitogen-activated protein kinase signaling pathways when oncogenic Ras
is overexpressed in NIH 3T3 cells (35). However, these experiments were
done under conditions in which the Ras or the Ras target is
overexpressed. In our mouse immortalized melanocytes system, we could
investigate these Ras signaling components in a normal cellular context
in response to chemokine stimulation.
Our earlier studies showed that MGSA/GRO-induced melanocyte
transformation depends on the ability of MGSA/GRO
to activate Ras
activation through the CXCR2 receptor (36). However, the downstream
components of the Ras-affected pathways in melanoma have not been fully
elucidated. Thus, we performed experiments to define the MGSA/GRO
intracellular signaling pathways in murine melanoma cells. Our data
show that MGSA/GRO
induces NF-
B activation as well as MEKK1,
MEK3/6, and p38 activity. This MGSA/GRO
induction of NF-
B is
dependent on activation of the Ras/MEKK/p38 cascade, based upon
experiments using dominant negative Ras, dominant negative MEKK1 or p38
inhibitors (SB202190 and SB203580). However, MGSA/GRO
activation of
NF-
B is independent of the MEK1/ERK/ELK and JNK pathways, because
MGSA/GRO
fails to induce activation of ERK/ELK and JNK, and the
inhibitor for MEK1 (PD98059) has no effect on MGSA/GRO
-enhanced
NF-
B activation. Finally, we report that this NF-
B activation is
required for MGSA/GRO
-induced melanocyte transformation.
 |
MATERIALS AND METHODS |
Cell Culture--
The nontransformed mouse immortalized
melanocyte cell line, parental melan-a (gift of Dr. Dorthea Bennett),
was cultured in Dulbecco's modified Eagle's medium supplemented with
50 units/ml penicillin, 50 µg/ml streptomycin, 3 mM
glutamine, 10% heat-inactivated fetal bovine serum (Life Technologies,
Inc.) and 200 nM
12-O-tetradecanoylphorbol-13-acetate (Sigma). The
V1,
3-14, Mel-a-6, E6A, L7A, and R8A clones
were cultured in the same medium supplemented with 800 µg/ml G418
(Sigma) as described previously (Ref. 11 and Table
I). The equal expression level of
MGSA/GRO
or CXCR2 receptor in these clones has been previously
verified (11, 36). The MEK1 inhibitor (PD98059), the p38 inhibitors
(SB202190 and SB203580), and the CXCR2 inhibitor (SB 225002)
(Calbiochem-Novabiochem) were prepared as a stock in
Me2SO (10 mM). The pertussis toxin was
purchased from Sigma. Purified recombinant human MGSA/GRO
(a kind
gift of Repligen Corp., Needham, MA) was used at 50 ng/ml.
Nuclear Extracts and Mobility Shift Assay--
Cells were lysed
with buffer (10 mM HEPES, 10 mM sodium
chloride, 1.5 mM magnesium chloride, 0.5 mM
dithiothreitol, 5 mM
-mercaptoethanol, 100 µM phenylmethylsulfonyl fluoride) with 0.5% Nonidet
P-40. The nuclear proteins were extracted from the nuclear pellets
using high salt extraction (25% glycerol, 0.2 mM EDTA, 20 mM HEPES, 1.5 mM magnesium chloride, 0.5 mM dithiothreitol, 10 mM potassium chloride,
and 240 mM sodium chloride) with protease inhibitors and
phosphatase inhibitors, as described previously (37). EMSA were
performed by incubating 0.25 ng of double-stranded oligonucleotide end-labeled with 32P by polynucleotide kinase (New England
Biolabs, Beverly, CA) and equal amounts of nuclear protein (5 µg) for
30 min at room temperature, as described previously (37). The binding
reaction mixtures were resolved in a 6% nondenaturing polyacrylamide
gel. Antibody supershift EMSA were performed by incubating nuclear extract proteins with antibodies (1 µg) against p65, p50, c-Rel, or
control antibody r-IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
at room temperature for 1 h before adding the labeled probe.
Whole Cell Extracts and Western Blot--
Whole cell extracts
were prepared from control, MGSA/GRO-treated parental melan-a cells,
and MGSA/GRO-expressing melan-a cells after serum-free starvation for
4 h. Western blots were performed following protocols provided by
Santa Cruz Biotechnology Inc. The cells were washed at 4 °C with 1×
phosphate-buffered saline and lysed in 0.25-0.5 ml of RIPA buffer (1×
phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate,
0.1% SDS) with protease inhibitor mixture tablets (Roche Molecular
Biochemicals). 50 µg of soluble protein was boiled, loaded, and
electrophoretically separated on a 10% SDS-PAGE and then
electrophoretically transferred to a 0.45-µm nitrocellulose membrane
(BIO-RAD, Hercules, CA). The membrane was blocked with 5% dry milk in
TBS-T buffer (10 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.05% Tween-20) for 1 h and then incubated for 12-16 h at
4 °C in a 1:200 dilution (1 µg/ml) of the anti-p-MEK3/6 (B-9)
antibody, the anti-p-p38 antibody (D-8), the anti-p-ERK antibody (E-4),
the anti-p-JNK antibody (G-7) (Santa Cruz Biotechnology, Inc.), or the
anti-p-ELK1 (9181) (New England BioLabs), respectively. Then the
membrane was incubated in a 1:3000 dilution of the appropriate
anti-mouse, anti-goat, or anti-rabbit immunoglobulin conjugated with
horseradish peroxidase (Roche Molecular Biochemicals) in TBS-T buffer
with 5% dry milk for 1 h at room temperature. The protein bands
were detected with the ECL Western blotting detection reagents
(Amersham Pharmacia Biotech) according to the manufacturer's
instructions. The blots were stripped and reprobed with anti-MEK3
(N-20) and MEK6 (N-19) antibodies, anti-p38 antibody (A-12), or
anti-ELK1 antibody (9182) (New England BioLabs).
Transient Transfection Assays--
Wild type M-Ras,
constitutively active M-Ras (G22V), and dominant negative M-Ras (S27N)
expression constructs and pBK-CMV expression vector were gifts from Dr.
Takeshi Endo. The dominant active MEKK1 and dominant negative MEKK1
(dN) expression constructs and pCMV5Myc expression vector were gifts
from Dr. Melanie Cobb. The NF-
B luciferase reporter gene construct
was constructed by inserting two copies of the NF-
B consensus DNA
binding sequence into the pGL-3 promoter vector (Promega, Madison, WI).
The pSV-
-galactosidase expression construct was obtained from
Promega. 4 × 105 cells (6-well plates) were
transiently cotransfected with 0.2 µg of NF-
B luciferase reporter
gene and 0.4 µg of expression constructs indicated in figures,
together with either 0.4 µg of pSV-
-galactosidase or pBL-CAT3
expression construct by the LipofectAMINE Plus reagent following
manufacturer's protocol (Life Technologies, Inc.). Three hours later,
the cells were incubated with complete medium at 37 °C/5%
CO2. The cells were treated with inhibitors, MGSA/GRO
,
or both, after serum-free starving for 4 h. Two days later, the
cells were washed with cold phosphate-buffered saline and lysed in 1×
reporter lysis buffer (Promega) for 15 min at room temperature, and the
lysate was cleared by centrifugation. The luciferase,
-galactosidase, or CAT activity was measured according to standard
protocols (Promega) using a Monolight 2010 luminometer (Analytical
Luminescence Laboratory, San Diego, CA) or Beckman LS 3801. Transfection efficiency was monitored by assaying for CAT or
-galactosidase activity derived from a cotransfected expression
constructs. All p values were obtained by using the Student's two-tailed t test.
Immune Complex Kinase Assays--
Whole cell extracts were
prepared from control and MGSA/GRO
-treated parental melan-a cells
after serum-free starvation for 4 h. MEKK kinase assays were
performed as described in the manufacturer's protocol (Upstate
Biotechnology, Lake Placid, NY). 400 µg of protein of each whole cell
extract was immunoprecipitated with MEKK1 antibody (Santa Cruz).
Immunoprecipitated MEKK1 activity was assayed using the MEKK1
substrate, GST fusion protein MEK4 (inactivated) (Upstate Biotechnology). Kinase reactions were initiated by addition of 2 µg
of GST-MEK4 and kinase buffer containing 500 µM cold ATP and 10 µCi of [
-32P]ATP. Reactions were incubated
for 30 min at 30 °C and terminated by the addition of an equal
volume of 2× SDS loading buffer followed by boiling for 5 min.
Phosphorylated proteins were resolved by 10% SDS-PAGE and transferred
to a 0.45-µm nitrocellulose membrane (Bio-Rad). The phosphorylated
bands were visualized by autoradiography. The blot was probed with
MEKK1 antibody to monitor equal loading of MEKK1.
Transformation Assays--
Focus formation assays were performed
in triplicate in three independent experiments. Mel-a-6 cells were
cotransfected with 0.2 µg of a puromycin resistant vector (pBABE) and
either 2 µg of pCMV4 (vector) or I
B-
(wild type or
N) by
LipofectAMINE Plus reagent (Life Technologies, Inc.). The cells were
cultured in 5% fetal bovine serum/Dulbecco's modified Eagle's medium
with 0.8 mg/ml G418 and 0.5 mg/ml puromycin, and the foci of
transformed cells were counted 18 days after transfection. All
p values were obtained by using the Student's two-tailed
t test.
 |
RESULTS |
MGSA/GRO Increases NF-
B Activation--
Earlier results showed
high constitutive nuclear NF-
B activity in human Hs294T melanoma
cells as compared with normal retinal pigment epithelial cells (37).
Hs294T cells secrete a high level of MGSA/GRO
protein, whereas
retinal pigment epithelial cells cells do not express MGSA/GRO
protein (37). Here, we investigated whether MGSA/GRO
induces the
activation of NF-
B in mouse melanocytes (parental melan-a cells).
First, we performed EMSA to evaluate DNA binding activity in parental
melan-a cells treated with MGSA/GRO
for the indicated times by using
a 32P-labeled consensus NF-
B DNA binding sequence as the
probe. As seen in Fig. 1A
(left panel), there are two shifted NF-
B nuclear complexes bound to the probe, and MGSA/GRO
induces an increase in
the formation of these complexes over time (10-120 min). Based on
supershift EMSA as shown in Fig. 1B, the lower band
consisted of the p50 homodimer, whereas the upper band is the p65/p50
heterodimer. The antibody to c-Rel did not affect the mobility of the
NF-
B complexes (Fig. 1B). When the ELR motif of
MGSA/GRO
is mutated, the ligand exhibits marked reduction in its
affinity for receptor, CXCR2 (38). EMSA analysis of nuclear extracts
from MGSA/GRO
-expressing clones (Mel-a-6), and each of three ELR
motif mutant forms of MGSA/GRO
-expressing clones (E6A/ELR, L7A/ELR
and R8A/ELR) revealed that overexpression of MGSA/GRO
increased the
NF-
B p65/p50 heterodimer binding activity, whereas all three ELR
motif mutant forms failed to enhance NF-
B binding activity (Fig.
1A, right panel). The V1 clone
exhibited higher basal p50 homodimer binding activity than the parental
melan-a cells. This low level of p50 homodimer NF-
B in
V1 cells correlates with a slightly greater constitutive secretion of MIP-2 (138 pg/ml) and KC (49 pg/ml) in these cells, as
compared with the parental melan-a cells (MIP-2 (0 pg/ml) and KC (21 pg/ml); Table II). Taken together,
our data suggest that exogenous addition or overexpression of
MGSA/GRO
in the parental melan-a cells enhances NF-
B binding
activity, and the ELR motif of MGSA/GRO
is required for this
induction.

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Fig. 1.
MGSA/GRO increases
NF- B activation. A, NF- B
binding activity. In the left panel, the radiolabeled
double-stranded NF- B consensus oligonucleotide
(AGTTGAGGGGACTTTCCCAGGC) was mixed with nuclear extracts (5 µg)
prepared from parental melan-a cells untreated (lane 1) or
50 ng/ml MGSA/GRO for the indicated times (lanes 2-4).
Then EMSA was performed as described under "Experimental
Procedures." The positions of the NF- B subunits are indicated. In
the right panel, EMSA was performed on the nuclear extracts
from the following melan-a clones: V1 (lane 1),
Mel-a-6 (lane 2), E6A (lane 3), L7A (lane
4), and R8A (lane 5). B, NF- B supershift.
Nuclear extract (5 µg) from the parental melan-a cells treated with
50 ng/ml MGSA/GRO for 120 min were preincubated with 1 µg of
rabbit IgG (lane 2), -p65 (lane 3), -p50
(lane 4), -c-Rel (lane 5), -p50 and -p65
(lane 6), -p65 and -c-Rel (lane 7), -p50
and -c-Rel (lane 8), or -p50, p65 and -c-Rel
(lane 9) at room temperature for 1 h. Then EMSA was
performed as described for A. C, NF- B
transactivation. Parental melan-a, V1, Mel-a-6, E6A, L7A,
or R8A cells were transiently cotransfected with 0.2 µg of NF- B
luciferase reporter gene and 0.4 µg of pSV- -galactosidase
expression construct. Luciferase and -galactosidase activity were
measured 48 h later. The relative luciferase activity represents
the luciferase activity normalized by -galactosidase activity. The
bar graph shows a mean (± S.E.) of fold induction from four
independent transfections (the relative luciferase activity of
V1 cells was arbitrarily set to 1, all other activities
were given relative to this standard).
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Table II
Basal chemokine expression, ras activity, and NF- B activity in
different clones
Endogenous MIP-2 and KC levels were determined by enzyme-linked
immunosorbent assay from conditioned medium collected from parental
melan-a cells or V1 cells grown in the absence of serum. The
mean result from two individual assays performed in duplicate are shown
in ng/ml. The basal level of activated Ras in parental and V1
cells is shown from our previous work (36). NF- B activation was
determined as described under "Materials and Methods."
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Second, we tested the transactivation of these MGSA/GRO
-enhanced
NF-
B complexes. The cells were cotransfected with a luciferase reporter gene driven by the SV40 promoter containing two copies of the
NF-
B consensus element and pSV-
-galactosidase expression construct for internal control. MGSA/GRO
-expressing cells (Mel-a-6) exhibited significantly increased NF-
B transactivation (2.5-fold, p < 0.01) compared with the V1 cell.
Constitutive expression of ELR mutant forms of MGSA/GRO
failed to
increase the NF-
B transactivation (Fig. 1C). The
V1 cells exhibited 2-fold higher basal NF-
B
transactivation than the parental cells (Fig. 1C). The
observation is consistent with the higher NF-
B binding activity in
V1 cells than in the parental melan-a cells. To
further confirm that the high basal NF-
B activation in
V1 cells is mediated by CXCR2, we tested the ability of a
selective CXCR2 inhibitor, SB 225002 (39), as well as the ability of
pertussis toxin (PTx) to inhibit this high basal NF-
B activation.
Signal transduction mediated by ligand binding of CXCR2 has been shown
to be dependent on the interaction of the receptor with the
PTx-sensitive Gi proteins in neutrophils and 293 cells
stably transfected to express CXCR2 (40). V1 cells were
cotransfected with an NF-
B-dependent luciferase reporter and the pSV-
-galactosidase expression construct and then treated with highly specific chemical inhibitors for CXCR2 (SB225002). The
results showed that NF-
B transactivation enhanced by endogenous MIP-2 and KC is blocked 29, 35, or 49% by these inhibitors at 10, 50, or 100 nM, respectively (Table
III). Similarly, PTx inhibits this basal
NF-
B transactivation by 32, 52, or 63% at 0.5, 1, or 2 µg/ml,
respectively (Table III). These data support the concept that the
higher basal NF-
B transactivation in V1 cells is
mediated by CXCR2 and PTx-sensitive Gi proteins. Taken
together, our data demonstrate that MGSA/GRO
enhances NF-
B
activation. This activation requires the ELR motif, suggesting the
ligand must activate its receptor, CXCR2, on melanocytes.
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Table III
A selective CXCR2 inhibitor, SB 225002, or PTx inhibits the high basal
NF- B transactivation in VI cells
The mean results from two individual experiments performed in duplicate
are shown in fold induction.
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Ras Mediates MGSA/GRO
-enhanced NF-
B Activation--
Our
earlier results showed that an early response to MGSA/GRO
involves
activation of Ras, whereas an increase in the expression of Ras protein
occurs as a later event (36). We postulated that the MGSA/GRO
up-regulation of Ras may correlate with MGSA/GRO
-induced NF-
B
activation. To determine whether Ras could directly induce NF-
B
activation in melanocytes, V1 cells were cotransfected with the NF-
B luciferase reporter construct and an M-Ras expression construct (either wild type, constitutively active, or control vector
pBK-CMV). The expression of wild type M-Ras or a constitutively active
M-Ras (G22V) increased basal NF-
B transactivation by 2.8- (p < 0.001) and 4.9-fold (p < 0.001)
in V1 cells, respectively (see Fig. 4C). Because the
expression of wild type M-Ras or dominant active M-Ras in melan-a
clones did not affect the activation of the IP-10 promoter CAT reporter
construct (data not shown), the effects of Ras on NF-
B are not the
result of a general enhancement of metabolic activity or transcription
activity. To understand why the expression of wild type M-Ras increased
NF-
B activation even in the absence of exogenous MGSA/GRO
, we
examined the basal level of activated Ras in the V1 cells
transfected with wild type M-Ras, using the GST-Raf Ras-binding domain,
which precipitates only activated Ras (Ras-GTP but not Ras-GDP) (36).
Ras transfected V1 cells exhibited 3-fold higher level of
activated Ras than the V1 cells transfected with PBK-CMV
vector alone (Table II). We postulate that the continuous exposure to
endogenous MIP-2 and KC produced by the V1 cells is
sufficient to activate Ras and enhance basal NF-
B transactivation in
V1 cells. We have previously demonstrated that exogenous
addition of MGSA/GRO
to parental melan-a cells induced Ras
activation (36). The extremely low level of endogenous MIP-2 and KC
secretion in the parental melan-a cells was paralleled by little, if
any, endogenous Ras activity in these cells (Table II). To directly
evaluate whether Ras mediates MGSA/GRO-induced NF-
B activation, we
examined the effect of dominant negative M-Ras (S27N) on
MGSA/GRO-induced NF-
B transactivation. The NF-
B transactivation
enhanced by expression of MGSA/GRO
in Mel-a-6 was reduced by the
dominant negative M-Ras (S27N) in a plasmid
concentration-dependent manner. With a dominant negative M-Ras (S27N) plasmid concentration of 0.4, 0.8, or 1.2 µg in the transfection reaction, the basal NF-
B activation in V1
cells was decreased to 83, 65, or 41% of the control in V1
cells transfected with pBK-CMV vector only, respectively (Fig.
2A). Similarly, the expression
of the dominant negative M-Ras directly blocked NF-
B activation
stimulated by exogenous MGSA/GRO
treatment after transfection in
V1 cells. (Fig. 2B). In parallel, the expression
of the dominant negative M-Ras in parental melan-a cells inhibited the
exogenous MGSA/GRO
-induced NF-
B activation (data not shown). The
expression of dominant negative M-Ras in Mel-a-6 cells did not affect
the activation of the IP-10 CAT reporter (data not shown),
demonstrating that the effects of dominant negative M-Ras on NF-
B
activation are not nonspecific. Taken together, these data demonstrate
that Ras mediates MGSA/GRO-enhanced NF-
B activation.

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Fig. 2.
Ras mediates
MGSA/GRO -enhanced
NF- B activation. A, Dominant
negative M-Ras blocks NF- B transactivation enhanced by expression of
MGSA/GRO. V1 cells (empty bars) or
MGSA/GRO -expressing cells (Mel-a-6; solid bar) were
cotransfected with 0.2 µg NF- B luciferase reporter and indicated
amount of pBK-CMV vector or/and the dominant negative M-Ras, together
with 0.2 µg of pBL-CAT3 construct. Luciferase and CAT
activity were measured 48 h later. The relative luciferase
activity represents the luciferase activity normalized by CAT activity.
The results are reported as a mean (± S.E.) of relative luciferase
activity (the luciferase activity normalized by CAT activity) from
three independent experiments. B, dominant negative M-Ras
blocks MGSA/GRO -induced NF- B transactivation. V1
cells were cotransfected as described for A. After
transfection, cells were either untreated (control) or treated with 50 ng/ml MGSA/GRO for 48 h. The results are reported as a mean (± S.E.) of the relative luciferase activity (the luciferase activity
normalized by CAT activity) from three independent experiments.
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MGSA/GRO
Increases Activation of MEKK1--
Because MGSA/GRO
triggered Ras activation and Ras regulates the MEKK1 pathway, it was of
interest to determine whether MGSA/GRO
could activate the MEKK1
pathway in mouse melanocytes (parental melan-a cells). To address the
question of whether MGSA/GRO
increases the endogenous MEKK1 kinase
activity, immune complex kinase assays were performed. The results of
these assays showed that MGSA/GRO
increased the phosphorylation of
MEK4, which is one of the substrates of MEKK1 (Fig.
3A). The time course of
MGSA/GRO
induction of MEKK1 activation is comparable with that of
MGSA/GRO
activation of Ras. The maximal Ras activation occurs at 10 min and lasts 120 min (36).

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Fig. 3.
MGSA/GRO increases
MEKK1 activity. A, MEKK1 kinase activity. Parental
melan-a cells were either untreated or stimulated with 50 ng/ml
MGSA/GRO for the indicated times after serum starvation for 4 h. Endogenous MEKK1 was immunoprecipitated from whole cell extracts.
MEKK1 activity was determined by an immunocomplex kinase assay using
inactive MEK4 as a substrate as described under "Experimental
Procedures." Phosphorylated proteins were resolved on 10% SDS-PAGE
and transferred to a nitrocellulose membrane. The phosphorylated MEK4
bands were visualized by autoradiography (top panel). The
blot was probed with MEKK1 antibody to monitor equal loading of MEKK1
(middle panel). This figure is representative of three
different experiments. The bar graph (bottom panel)
represents the mean fold-induction (± S.E.) of the relative band
density of MEK4 (the band density of MEK4 were normalized by the MEKK1
band density) quantitated by densitometer from three independent
experiments. B, MEK3/6 phosphorylation. Parental melan-a
cells were treated as in A. Whole cell extracts were
subjected to 10% SDS-PAGE and immunoblotting with anti-p-MEK3/6
(top panel). The blot was stripped and reprobed with
anti-MEK3/6 to assess equal loading of lysates (middle
panel). This figure is representative of three different
experiments. The bar graph (bottom panel) represents the
mean fold-induction (± S.E.) of the relative band density of
phosphorylated MEK3/6 (the band density of phosphorylated MEK3/6 were
normalized by the MEK3/6 band density) quantitated by densitometer from
three independent experiments.
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MEKs Are Downstream Targets of MEKKs--
Previous studies have
identified MEK3, MEK4, or MEK6 as a cellular kinase directly regulating
p38 activity. To determine whether MGSA/GRO
could activate
endogenous MEK3/6 activity, Western blots were performed to detect
Ser-189 and Ser-207 phosphorylated MEK3/6 using a specific
anti-phosphorylated MEK3/6 antibody (B-9) (Santa Cruz). We observed
that MGSA/GRO
enhanced the phosphorylation of endogenous MEK3/6
(Fig. 3B). MGSA/GRO
activation of MEK3/6 follows the
activation profiles of MEKK1. These data indicate that MEKK1 and its
substrates MEK3/4/6 could be involved in MGSA/GRO
signal pathways.
MEKK1 Mediates MGSA/GRO
-enhanced NF-
B Activation--
The
above observation indicated that MEKK1 is involved in MGSA/GRO
signal pathways. To determine whether MEKK1 could induce NF-
B
activation in melanocytes, V1 cells were cotransfected with an expression vector containing the constitutively active MEKK1 construct together with the NF-
B luciferase reporter and
pSV-
-galactosidase expression construct. The expression of a
constitutively active MEKK1 (dominant active) increased NF-
B
activity by 6-fold (p < 0.01) (Fig.
4A, left panel). To
examine whether MEKK1 mediates MGSA/GRO
-enhanced NF-
B activation,
we examined the effect of dominant negative MEKK1 (dN) on
MGSA/GRO
-induced NF-
B transactivation. The NF-
B
transactivation enhanced by the expression of MGSA/GRO
in Mel-a-6
cells was reduced by the dominant negative MEKK1 (dN) to 74, 46, or
27% at MEKK1 (dN) plasmid concentration of 0.4, 0.8, or 1.2 µg in
the transfection reaction, respectively, whereas the basal NF-
B
activation in V1 cells was reduced by the dominant negative
MEKK1 (dN) to 37% or 30% at MEKK1 (dN) plasmid concentration of 0.8 or 1.2 µg in the transfection reaction, respectively (Fig. 4A, right panel). Similarly, the expression of
dominant negative MEKK1 (dN) can directly block NF-
B activation
stimulated by exogenous MGSA/GRO
treatment after the transfection in
parental melan-a cells by 78% (p < 0.01) (Fig.
4B). These results are similar to those obtained in
MGSA/GRO
-expressing clones. These data demonstrate that MEKK1
mediates MGSA/GRO
-enhanced NF-
B activation.

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Fig. 4.
MEKK1 mediates
MGSA/GRO -enhanced
NF- B activation. A, Dominant
active MEKK1 increases NF- B transactivation in V1,
whereas dominant negative MEKK1 blocks NF- B transactivation enhanced
by expression of MGSA/GRO . In the left panel,
V1 cells were cotransfected with 0.2 µg of NF- B
luciferase reporter and 0.4 µg of the indicated construct (either
pCMV5Myc vector or dominant active MEKK1), together with 0.4 µg of
pSV- -galactosidase expression construct. In the right
panel, V1 cells or MGSA/GRO -expressing cells
(Mel-a-6) were cotransfected with 0.2 µg of NF- B luciferase
reporter and indicated amount of constructs (either pCMV5Myc vector
or/and the dominant negative MEKK1), together with 0.2 µg of
pSV- -galactosidase expression construct. The luciferase activity and
-galactosidase activity were measured. The results are reported as
the means ± S.E. of the relative luciferase activity (the
absolute luciferase activity of sample was normalized by
-galactosidae activity) from three independent experiments.
B, dominant negative MEKK1 blocks exogenous
MGSA/GRO -induced NF- B transactivation. Parental melan-a cells
were cotransfected as described for A. After transfection,
cells were either untreated or treated with 50 ng/ml MGSA/GRO for
48 h. The results are reported as the means ± S.E. of the
relative luciferase activity (the absolute luciferase activity of
sample was normalized by -galactosidase activity) from three
independent experiments. C, dominant negative MEKK1 blocks
Ras-induced NF- B transactivation. V1 cells were
cotransfected with 0.2 µg of NF- B luciferase reporter and 0.4 µg
of the indicated constructs, together with 0.4 µg of
pSV- -galactosidase expression construct. The luciferase activity and
-galactosidase activity were measured. The relative luciferase
activity represents the luciferase activity normalized by
-galactosidase activity. The results are reported as a the
means ± S.E. of fold induction considering 1 as the relative
luciferase activity of the cells transfected with the corresponding
empty vectors from three independent experiments.
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|
To determine whether MEKK1 is a downstream effector of Ras in the
signal pathway of MGSA/GRO
-enhanced NF-
B activation, we analyzed
the effect of inhibition of MEKK1 on Ras induction of NF-
B
activation by using dominant negative MEKK1 (dN). V1 cells were cotransfected with the dominant negative MEKK1 expression construct and either the wild type M-Ras or the dominant active M-Ras
(G22V) expression construct, together with the NF-
B luciferase reporter and pSV-
-galactosidase expression constructs. The
expression of dominant negative MEKK1 (dN) blocks both wild type and
dominant active M-Ras activation of NF-
B by 67% (p < 0.02) and 95% (p < 0.03), respectively (the
luciferase activity in cells cotransfected with M-Ras and MEKK1
constructs was compared with the cells cotransfected with M-Ras and
pCMV5Myc empty vector) (Fig. 4C). This observation was
reproduced in parental melan-a cells (data not shown). These data
demonstrate that MEKK1 is a downstream target of Ras in MGSA/GRO
activation of NF-
B.
p38 MAP Kinase Is Involved in MGSA/GRO
-enhanced NF-
B
Activation--
MEKK1 phosphorylates and activates MEK3, MEK4, and
MEK6 kinases in the p38 pathway in vitro (24-27).
Activation of p38 requires phosphorylation on Thr-180 and Tyr-182 (41,
42) and can be assayed directly using specific antibodies to the
phosphorylated form of p38. To examine whether MGSA/GRO
induces p38
phosphorylation, Western blots were performed by using whole cell
extracts from parental melan-a cells treated with MGSA/GRO
for the
indicated times. As seen in Fig.
5A, MGSA/GRO
enhanced p38
phosphorylation at 30 min (Fig. 5A, left panel).
The results revealed that p38 is involved in MGSA/GRO signal pathways
and that the profile of p38 activation is consistent with that of
MEK3/6 activation. In parallel, Western blots analysis of
phosphorylated p38 from V1, MGSA/GRO
-expressing clones
(Mel-a-6), and ELR motif mutant MGSA/GRO
-expressing clones (R8A)
revealed that expression of MGSA/GRO
increased the p38
phosphorylation, whereas the ELR motif mutant form failed to enhance
this phosphorylation (Fig. 5A, right panel).

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Fig. 5.
p38, but not ERK and JNK, is involved in
MGSA/GRO -enhanced
NF- B activation. A,
MGSA/GRO increases p38 phosphorylation. Left panel,
parental melan-a cells were treated as in Fig. 3A. Whole
cell extracts were subjected to 10% SDS-PAGE and immunoblotting with
anti-p-p38 (top panel). The blot was stripped and reprobed
with anti-p-38 to assess equal loading of lysates (middle
panel). This figure is representative of three different
experiments. The bar graph (bottom panel) represents the
mean fold induction (± S.E.) of the relative band density of
phosphorylated p38 (the band density of phosphorylated p38 were
normalized by the p38 band density) quantitated by densitometer from
three independent experiments. Right panel, whole cell
extracts from V1, Mel-a-6, and R8A clones were subjected to
10% SDS-PAGE and immunoblotting with anti-p-p38 (top
panel). The blot was stripped and reprobed with anti-p38 to assess
equal loading of lysates (bottom panel). This figure is
representative of two different experiments. B, p38
inhibitors, but not MEK1 inhibitor, block NF- B transactivation
enhanced by expression of MGSA/GRO . Left panel,
V1 or MGSA/GRO -expressing cells (Mel-a-6) were
cotransfected as in Fig. 1C. After transfection, the cells
were treated with either the solvent control (Me2SO) or the
indicated concentrations of PD98059, SB202190, or SB203580 for 24 h. Right panel, V1 or Mel-a-6 cells were
transiently cotransfected with 0.2 µg of AP-1 luciferase reporter
gene and pSV- -galactosidase expression construct. After
transfection, the cells were treated as in the above panel.
C, p38 inhibitors, but not MEK1 inhibitor, block exogenous
MGSA/GRO -enhanced NF- B transactivation. Parental melan-a cells
were transiently cotransfected with 0.2 µg of NF- B luciferase
reporter gene and 0.4 µg of pSV- -galactosidase expression
construct. The cells were pretreated with either the solvent control
(Me2SO) or the indicated concentrations of PD98059 or
SB202190 for 2 h and then stimulated with MGSA/GRO for 24 h. Luciferase activity and -galactosidase activity were measured.
The results are reported as the means (± S.E.) of the relative
luciferase activity (the absolute luciferase activity of sample was
normalized to -galactosidase activity) from three independent
experiments. D, MGSA/GRO failed to increase ERK, JNK, and
ELK phosphorylation. Parental melan-a cells were treated as in
A. Whole cell extracts were subjected to 10% SDS-PAGE and
immunoblotting with anti-P-ERK (top panel), anti-P-JNK
(second panel), or anti-P-ELK (third panel). The
blot was stripped and reprobed with anti-ELK to assess equal loading of
lysates (bottom panel). This figure is representative of
three different experiments.
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|
To evaluate the role of p38 in regulating MGSA/GRO
-enhanced NF-
B
activation in vivo, MGSA/GRO
-expressing cells (Mel-a-6) were cotransfected with an NF-
B-dependent luciferase
reporter and the pSV-
-galactosidase expression construct and then
treated with highly specific chemical inhibitors for p38 (SB202190 and SB203580) (41) at the indicated concentrations. The results showed that
NF-
B transactivation enhanced by expression of MGSA/GRO
is
blocked 53% (p < 0.02), 70% (p < 0.01), or 100% (p < 0.01) by these inhibitors at 5, 15, or 30 µM, respectively (Fig. 5B, left panel). In contrast, these inhibitors have no effect on
AP-1 activation enhanced by expression of MGSA/GRO
(p > 0.2) (Fig. 5B, right
panel). Similarly, parental melan-a cells were cotransfected with
an NF-
B-dependent luciferase reporter and the
pSV-
-galactosidase expression construct, pretreated with SB202190 or
PD98059, and then stimulated by exogenous MGSA/GRO
. The inhibition
of the p38 kinase with SB202190 reduced NF-
B transactivation to near control levels at 30 µM (p < 0.01) (Fig.
5C). The concentration of inhibitors blocking activation of
NF-
B is similar to that needed for inhibition of p38 kinase activity
induced by G-CSF in BaF3-WT cells (43). The observation that p38
inhibitors abrogate MGSA/GRO
stimulation of NF-
B suggests that
p38 is involved in MGSA/GRO
activation of NF-
B.
The MEK1/ERK Kinase Cascade Is Not Essential for NF-
B Activation
by MGSA/GRO
--
When overexpressed, MEKK1 also activates the ERK
and JNK pathways (44, 45). MEKK1 phosphorylates and activates MEK1,
which leads to activation of ERK1/2 (23). We therefore asked whether NF-
B activation by MGSA/GRO
is involved in this MEK1-ERK cascade. First, we examined whether MGSA/GRO
regulates ERK activity in parental melan-a cells. Phosphorylation of ERK at Tyr-204 is involved in activation of ERK. MGSA/GRO
failed to enhance the phosphorylation of ERK in parental melan-a cells (Fig. 5D). A similar
approach, using an antibody that only recognizes phosporylated
(activated) ELK on Western blots showed that MGSA/GRO
was unable to
increase the phosphorylation of ELK, a known target of the MEK1-ERK
pathway (Fig. 5D). MGSA/GRO
also failed to enhance JNK
activation (Fig. 5D). These results suggest that the
MEK1-ERK cascade is not critical for MGSA/GRO
-enhanced NF-
B
activation in melan-a cells.
To further explore this question, we tested the effect of the MEK1
inhibitor, PD98059. The inhibition of MEK1 by PD98059 selectively blocks ERK activation at 10-50 µM in many systems (46).
PD98059 at 25 or 50 µM was added to MGSA/GRO
expressing cells (Mel-a-6) cotransfected with an
NF-
B-dependent luciferase reporter and pSV-
-galactosidase expression construct, and the luciferase and
-galactosidase activity were analyzed. Addition of PD98059 at 50 µM did not decrease basal NF-
B transactivation (Fig.
5B). Likewise, the PD98059 at 50 µM did not
reduce MGSA/GRO
-stimulated NF-
B-dependent reporter
activity in parental melan-a cells (Fig. 5C). Taken
together, these data suggest that MGSA/GRO
-enhanced NF-
B
activation is independent of the MEK1-ERK and JNK cascades.
Inhibition of NF-
B Activation Blocks MGSA/GRO
-induced
Melanocyte Transformation--
The observation that MGSA/GRO
can
enhance NF-
B activation prompted us to analyze whether NF-
B
activation is critical for cell transformation by MGSA/GRO
. We
inhibited NF-
B activity with a super-repressor form of I
B-
(N-terminal deletion) that cannot be phosphorylated or degraded and
therefore blocks the nuclear translocation of the p65/p50 complex and
subsequent transactivation of NF-
B-responsive genes (47).
MGSA/GRO
-expressing Mel-a-6 cells transfected with the I
B-
expression construct (wild type or N-terminal deletion) exhibited less
focus formation than cells transfected with the vector in an in
vitro transformation assay (p < 0.001) (Fig.
6A). Because I
B-
(N-terminal deletion) inhibits NF-
B activity (p < 0.001) but not AP-1 activation (Fig. 6B), we conclude that
NF-
B activity is involved in MGSA/GRO
-induced melanocyte
transformation.

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Fig. 6.
NF- B is involved in
MGSA/GRO -induced cellular transformation.
A, left panel, MGSA/GRO -expressing cells
(Mel-a-6) were cotransfected with 0.2 µg of a puromycin resistant
vector (pBABE) and either 2 µg of pCMV4 (vector) or I B- (wild
type or N terminus deletion mutant). The cells were cultured in 5%
fetal bovine serum/Dulbecco's modified Eagle's medium with G418 (0.8 mg/ml) and puromycin (0.5 mg/ml), and the appearance of foci of
transformed cells was stained with crystal violet and counted 18 days
after transfection. This figure represents one of three independent
experiments with similar results each time. Right panel, the
bar graph represents the mean (± S.E.) of relative inhibition (the
colony numbers from cells transfected with I B- (wild type) or
I B- ( N) divided by the colony numbers from cells transfected
with vector) from three independent experiments performed in triplicate
(n = 9). B, I B- inhibits NF- B
activation but not AP-1 activation in Mel-a-6. MGSA/GRO -expressing
cells (Mel-a-6) were cotransfected with either 0.2 µg of the NF- B
luciferase reporter (left panel) or the AP-1 luciferase
reporter (right panel) and 0.4 µg of the indicated
construct (either empty vector or I B- expression construct),
together with 0.4 µg of pSV- -galactosidase expression construct.
Luciferase and -galactosidase activity were measured 48 h
later. The results are reported as the means (± S.E.) of the relative
luciferase activity (the absolute luciferase activity of sample was
normalized by -galactosidase activity) from three independent
experiments performed in duplicate (n = 6).
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DISCUSSION |
Proinflammatory cytokines such as interleukin-1 and tumor necrosis
factor-
rapidly activate NF-
B in most cell types through an
NIK/MEKK-IKK-I
B signal pathway (48, 49). These cytokines induce
NF-
B activation by modulating I
B phosphorylation, ubiquitination, and proteolytic degradation and by releasing functional NF-
B dimers
to translocate to the nucleus (50). Chemotactic factors, such as the
lipid mediator platelet-activating factor (51) and SDF-1
(18), are
also reported to activate NF-
B in leukocytes or murine pre-B cells,
respectively. However, the mechanism of chemokine receptor activation
of NF-
B is not clear. This is the first report to identify the
signaling pathway for MGSA/GRO
activation of NF-
B, which is
involved in melanocyte transformation. This activation utilizes a
signaling pathway different from those employed by tumor necrosis
factor-
, interleukin-1, or phorbol 12-myristate 13-acetate.
It is well known that tumors such as melanoma, breast cancer (52),
human head and neck carcinoma (53), and T cell lymphoma (54) have
elevated constitutive activation of NF-
B. We postulated that the
constitutive activation of NF-
B in melanoma cells could be in part
due to an MGSA/GRO
autocrine loop. In this paper, we have focused on
identifying the signaling components involved in MGSA/GRO
-enhanced
NF-
B activation. We show that either exogenous addition of
MGSA/GRO
or continuous expression of MGSA/GRO
in parental melan-a
cells enhances Ras, MEKK1, MEK3/6, p38, and NF-
B activity, but not
ERK, JNK, and ELK activity. Using activated and dominant negative forms
of kinases or kinase inhibitors, we have demonstrated here that
MGSA/GRO
activation of NF-
B is through a Ras-MEKK1-MEKs-p38
cascade. Moreover, this MGSA/GRO
-enhanced NF-
B activation is
required for melanocyte transformation.
In this study, we have used two control cells, V1 cells and
parental melan-a cells. The V1 clone is a better control
for MGSA/GRO
expressing stable clones than parental melan-a cells
because the stable clones have been selected to express the neomycin
resistance gene based upon survival in culture medium containing with
G418. To monitor effects of exogenous MGSA/GRO
on downstream
signals, the parental melan-a cells are the appropriate control,
because parental melan-a cells represent the whole population of the
melanocytes. However, in some experiments, such as Figs. 1
(A and C), 2B, and 4C, we
used both V1 cells and parental melan-a cells. We found that our observations in these experiments in V1 cells were
reproduced by the parental melan-a cells. In general, V1
cells have higher NF-
B basal activity than the parental melan-a
cells, although the V1 cells are derived from the parental
melan-a cells. Our data suggest that this higher basal NF-
B activity
correlates with the constitutive secretion of MIP-2 and KC by these cells.
Ras activation has been shown to contribute to melanoma tumor
development (55-57). Our earlier results showed that Ras is required for MGSA/GRO
-induced melanocyte transformation (36). Ras has also
been shown to interact physically with numbers of downstream targets,
such as Raf, Rac, MEKK1, and phosphatidylinositol 3-kinase, and to
activate several different signaling pathways. Using Ras effector
dominant mutants, which are deficient in specific effector function,
Webb et al. (58) reported that Ras-mediated transformation in NIH 3T3 cells can occur independently of the Raf-MEK1-ERK1/2 pathway. However, the downstream components of the Ras-affected pathways in melanoma have not been fully elucidated. In our present study, we show that MEKK1 is a downstream target of Ras in the murine
melanoma model used. But we cannot rule out the possibility that Rac is
also a component of this pathway, because Ras can activate Rac (59),
and Rac can regulate MEKK1 as well (45).
MEKK activates the protein kinase MEK, which activates ERK, JNK, or p38
(23-30). Once activated, MAP kinases translocate to the nucleus and
activate transcription factors. ERK and JNK are also involved in
activating NF-
B (60, 61), but it is unlikely that ERK and JNK are
involved in the MGSA/GRO
signal pathway in the melan-a clones
studied here based on the following observations: 1) MGSA/GRO
was
not able to increase ERK/ELK and JNK activation and 2) the inhibition
of ERK by the inhibitor of MEK1, PD98059, had no effect on
MGSA/GRO
-enhanced NF-
B activation. It could be argued that the
failure of MGSA/GRO
to enhance ERK/ELK and JNK activation in melan-a
cells could be in part due to the high basal activity of these kinases.
However, the MEK1 inhibitor PD98059 failed to block basal NF-
B
activation. This result is in agreement with a recent report showing
that Raf-mediated NF-
B activation is independent of the MEK1/ERK
cascade (62). Moreover, our findings here correspond well with the
observation that constitutively active components of the MAP kinase
pathways are involved in oncogenic transformation in NIH 3T3 cells,
whereas ERK1/2 activation is not required for this transformation event
(63). Thus, we conclude that MGSA/GRO
activation of NF-
B in mouse
melanocytes involves MEKK1/MEK activation followed by p38 MAP kinase activation.
The p38 kinase is known to regulate various transcription factors, such
as ATF-2 and NF-
B, which in turn modulate the transactivation capacity of the transcription enhancers (64-66). It has been reported that phosphorylation of NF-
B p65 strongly increases p65
transcriptional activity (67, 68). Numerous kinases, including IKK
and
, protein kinases C and A, and tyrosine kinases are implicated
in the regulation of NF-
B p65 transactivation. Moreover, recent data
connect transcriptional activity of NF-
B p65 with the versatile coactivator proteins p300 and CBP, ATF-2, TFIIB, and TBP (69-71). It
is possible that p38 kinase may regulate these coactivators to increase
NF-
B-dependent gene expression. We have not, however, determined whether NF-
B p65 is a direct target of p38 or is an indirect target of the p38-regulated kinase. Alternatively, p38 may
regulate these coactivators to facilitate NF-
B-dependent gene expression. Future studies will address the exact mechanism by
which p38 activates NF-
B.
Several lines of evidence suggest that NF-
B also plays an important
role in cellular transformation. The Rel family of transcription factors traditionally has been known to be oncogenic (72), and it was
recently demonstrated that NF-
B activity protects cells from Ras
transformation-associated apoptosis. It has been reported that a low
level of NF-
B activation (3-4 fold) is correlated with the
anti-apoptotic function. In contrast, a high level of NF-
B
activation failed to protect apoptosis (73). MGSA/GRO
induces a low
level of transactivation of NF-
B (2.5-4 fold) by enhancing p65/p50
heterodimer binding activity in parental melan-a cells or
MGSA/GRO
-expressing cells without inducing endogenous expression of
other NF-
B-regulated chemokines, such as MIP-2 and KC (data not
shown). The blocking of this NF-
B activation inhibits
MGSA/GRO
-induced melanocyte transformation. These results suggest
that a low level of NF-
B induction may not be sufficient to
up-regulate inflammatory gene expression but may be sufficient to
protect cells from Ras transformation-associated apoptosis. In support
of this premise is our observation that selective inhibition of NF-
B
activation in melanocytes by targeted overexpression of a
super-repressor form of I
B-
(
N) initiated M-Ras-induced apoptosis (data not shown). In conclusion, MGSA/GRO
-induced
melanocyte transformation critically depends on a low level of NF-
B
activation through the Ras/MEKK1/p38 cascade.