(Received for publication, September 25, 1995)
From the
Melanoma growth stimulatory activity (MGSA)/growth regulated
(GRO) and interleukin-8 (IL-8) are highly related chemokines that have
a causal role in melanoma progression. Expression of these chemokines
is similar in that both require the NF-B element and additional
regions such as the CAAT/enhancer binding protein (C/EBP) element of
the IL-8 promoter. The constitutive and cytokine IL-1-induced promoter
activity of the chemokine MGSA/GRO
in normal retinal pigment
epithelial and the Hs294T melanoma cells is partially regulated through
the NF-
B element, which binds both NF-
B p50 and RelA
(NF-
B p65) homodimers and heterodimers. Mutational analysis of the
MGSA/GRO
promoter reveals that, in addition to the NF-
B
element, the immediate upstream region (IUR) is necessary for basal
expression in retinal pigment epithelial and Hs294T cells. Gel mobility
shift and UV cross-linking analyses demonstrate that several
constitutive DNA binding proteins interact with the IUR. Although this
region has sequence similarity to the several transcription factor
elements including C/EBP, the IUR includes sequences that have no
similarity to previously identified enhancer regions. Furthermore, RelA
transactivates through either the NF-
B element or the IUR,
suggesting a putative interaction between NF-
B and this novel
complex.
Chemokines play a major role in mediation of inflammation and
wound healing. Melanoma growth stimulatory activity
(MGSA()/GRO) and IL-8, members of the C-X-C
chemokine family, are similar at both the structural and functional
level (chemokines reviewed in (1) ). These proteins share
several common receptors including the IL-8 A and B receptors and the
Duffy antigen receptor for chemokines
(DARC)(2, 3, 4, 5, 6, 7) .
Both MGSA/GRO and IL-8 are highly chemotactic for
neutrophils(8, 9, 10, 11, 12) .
Northern analyses and immunocytochemical studies have revealed that
MGSA/GRO and/or IL-8 are produced by several cell types including
endothelial cells(13, 14) ,
fibroblasts(8, 15, 16, 17) ,
keratinocytes(8, 18, 19, 20, 21) ,
and retinal pigment epithelial cells(22) .
MGSA/GRO
expression increases as melanocytes progress to malignant
melanoma(18, 23, 24) . Furthermore, both
MGSA/GRO and IL-8 serve as autocrine growth factors for several
melanoma cell
lines(9, 18, 25, 26, 27) .
Three MGSA/GRO genes have been identified (MGSA/GRO, -
, and
-
)(28, 29, 30, 31) . The
MGSA/GRO
form was purified from culture medium conditioned by
Hs294T human melanoma cells(16, 32, 33) , and
overexpression of MGSA/GRO
in immortalized mouse melanocytes
enabled these cells to form tumors in athymic nu/nu mice(34) .
We have shown in Hs294T cells that there is a high constitutive level
of transcription of the MGSA/GRO
gene that cannot be significantly
induced by the cytokines IL-1 and tumor necrosis factor
. In
contrast, IL-1 markedly increases MGSA/GRO
gene transcription in
normal retinal pigment epithelial (RPE) cells(35) .
The
human IL-8 and MGSA/GRO genes contain an NF-B element within their
enhancer regions that has been shown to be necessary for
transcriptional activation of these
chemokines(35, 36, 37) . Furthermore, IL-8
gene regulation also requires the C/EBP element adjacent to the
NF-
B element(36) . The C/EBP and NF-
B complexes
directly interact or cross-couple to further enhance IL-8 gene
transcription(38, 39, 40) . Although the
chemokine MGSA/GRO has an essential role in inflammation and tumor
progression, regulation of MGSA/GRO gene expression is not as well
understood as the closely related IL-8 gene.
In this work, we
demonstrate that in addition to the NF-B element, the immediate
upstream region (IUR) is necessary for basal and cytokine induced
expression of MGSA/GRO
in RPE cells. Likewise, basal MGSA/GRO
promoter activity within the Hs294T melanoma cells also requires this
region. However, unlike the IL-8 promoter, neither C/EBP
nor
C/EBP
recognize the similar region in the MGSA/GRO
promoter.
Furthermore, RelA (NF-
B p65) transactivates MGSA/GRO
transcription either directly through the NF-
B element or
indirectly through the adjacent IUR. We have identified a novel complex
that is constitutively bound to the IUR in both normal RPE and melanoma
cells. We propose that transcriptional regulation of the MGSA/GRO
gene involves multiple factors that recognize the NF-
B and
immediate surrounding regions.
Figure 1: Expression of MGSA/GRO and IL-8 mRNA in the Hs294T melanoma and RPE cells. Total RNA from unstimulated or 5 units/ml IL-1-stimulated RPE or Hs294T cells for the time (hours) indicated was analyzed by Northern blot as described under ``Materials and Methods.'' Identical blots were hybridized with specific cDNA probes for MGSA/GRO and IL-8. Equal loading was verified by subsequently hybridizing the blots with a cDNA probe for the constitutive mRNA cyclophilin (1B15).
Mukaida et al.(36) had shown that both the
NF-B and the adjacent C/EBP binding elements were required for
IL-1 and tumor necrosis factor
activation of IL-8 in a
fibrosarcoma cell line. Further work demonstrated that C/EBP proteins
bound to the IL-8 promoter and that NF-
B directly interacted with
the C/EBP complexes (38, 39) . Sequence analysis
between the IL-8 and MGSA/GRO
promoter region indicated an almost
identical NF-
B element and adjacent C/EBP-like region (Fig. 2). The MGSA/GRO
nucleotide sequence adjacent to the
NF-
B element contains several nucleotides that are conserved
between previously identified C/EBP enhancers and are essential for
C/EBP binding and subsequent transactivation for the IL-8, IL-6,
albumin gene (DE1), and the serum amyloid A
genes(36, 43, 51, 52) . The MGSA/GRO
region also has close homology with the human immunodeficiency virus
type I (HIV-1) LBP-1 binding site, which is necessary in addition to
the NF-
B and Sp1 for full transcriptional activation of the HIV-1
long terminal repeat (53, 54, 55, 56) (Fig. 2).
This sequence was of interest since we have recently demonstrated that
basal MGSA/GRO
promoter activity required both NF-
B- and
Sp1-related complexes bound within the immediate promoter(57) .
Figure 2:
Sequence comparison of
MGSA/GRO-97/-62 and IL-8-100/-65 with
several consensus transcription factor elements. Sequence comparison of
MGSA/GRO
-97/-62 and IL-8-100/-65 with
several consensus transcription factor elements. The nucleotide
sequences of the MGSA/GRO
-97/-62 and IL-8
-100/-65 promoter regions containing the NF-
B element
are shown. The shaded boxes represent the NF-
B element
and either the NF-IL6 or the IUR in the IL-8 and MGSA/GRO
promoter, respectively. Nucleotides that are not conserved between the
indicated MGSA/GRO
promoter fragment and each consensus site are
indicated by dots. The consensus sequences of C/EBP binding
sites are IL-6(43) , IL-8(36) , albumin DE
1(51) , and serum amyloid A (SAA3) (52) . The consensus
RelA sequences are NF-
B p65 consensus(62) , IL-8 (36) , immunoglobulin
light chain enhancer (IgG
)(63) . The HIV long terminal repeat LBP-1 binding site
sequence is
included(53, 54) .
Figure 3:
IUR is required for MGSA/GRO promoter
activity in RPE and Hs294T cells. IUR is required for MGSA/GRO
promoter activity in RPE and Hs294T cells. RPE (A) or Hs294T (B) cells were co-transfected with 10 µg of the indicated
MGSA
350/CAT construct and 2 µg of pCMVhGH as described under
``Materials and Methods.'' Approximately 24 h after
transfection, the cells were either unstimulated (solid bars)
or stimulated with 5 units/ml IL-1 for 24 h (stripedbars) prior to collection. Results for RPE cell
transfection are expressed as -fold activation over cells transfected
with MGSA
350/CAT alone with no treatment. -Fold induction,
standard deviation of error, and number of separate transfections were
MGSA
350/CAT unstimulated (1.00) IL-1 (5.18 ± 1.65) (n = 5), mutant IUR MGSA
350/CAT unstimulated (0.26
± 0.18) IL-1 (0.46 ± 0.26) (n = 4),
mutant NF-
B MGSA
350/CAT unstimulated (0.40 ± 0.28)
IL-1 (0.41 ± 0.18) (n = 5), and double mutant
MGSA
350/CAT unstimulated (0.04 ± 0.02) IL-1 (0.05 ±
0.03) (n = 3). Results for basal promoter activity in
the Hs294T cells are expressed as percent CAT conversion. Percent
conversions and standard error from three separate transfections were
MGSA
350/CAT wild-type (7.22 ± 1.19), mutant IUR (0.09
± 0.07), and mutant NF-
B (0.70 ±
0.28).
Figure 4:
Characterization of nuclear proteins that
bind to the MGSA/GRO -97/-62 region. Characterization
of nuclear proteins that bind to the MGSA/GRO
-97/-62
region. Nuclear extracts (5 µg) from unstimulated (NT) or
IL-1 stimulated (IL-1) RPE cells were incubated 20
min at room temperature with either labeled wild-type (WT),
mutant IUR (m.IUR), mutant NF-
B (m.
B) or
double mutant (m.IUR+m.
B)
MGSA
-97/-62 oligonucleotides. Preimmune (PI)
antisera or antisera to NF-
B p50, NF-
B p52, RelA, or
C/EBP
were incubated with extracts prior to addition of labeled
probe. The resulting protein-DNA complexes were separated on 0.5
TBE polyacrylamide gels. The NF-
B p50/RelA heterodimer and
RelA complexes are indicated.
Figure 5:
Characterization of C/EBP-like elements in
the MGSA/GRO and IL-8 promoter. Characterization of C/EBP-like
elements in the MGSA/GRO
and IL-8 promoter. Recombinant C/EBP
protein was incubated with 100 pg of
P-labeled NF-IL6
oligonucleotide probe (40,000 cpm) as described under ``Materials
and Methods.'' Unlabeled competitor oligonucleotide probes NF-IL6,
MGSA
-97/-62, and IL-8-101/-63 were preincubated
15 min prior to the addition of labeled probe. The -fold excess of each
unlabeled competitor DNA is indicated. The resulting DNA-protein
complexes were separated on 0.25
TBE polyacrylamide gels.
Quantitation of bound protein-DNA complexes was done using the software
program IQ3.29 for the PhosphorImager (Molecular
Dynamics).
Figure 6:
Characterization of IUR constitutive
binding complexes. A, cytoplasmic extracts (lane 1)
or nuclear extracts from unstimulated (NT, lane 4) or
IL-1 stimulated (IL-1, lanes 2, 5-14) RPE cells were incubated in the presence of
labeled MGSA 2xIUR oligonucleotide. Unlabeled competitor probes
(50-fold excess) included IL-8 (lane 6), wild-type, and mutant
MGSA
-97/-62 and MGSA
-97/-78
oligonucleotides (lanes 7-14) (see Table 1for
sequences). B, nuclear extracts from IL-1-stimulated RPE cells
were preincubated with preimmune antisera (PI) (lane
2) or antisera to RelA, C/EBP
, and LBP (lanes
3-5) 20 min prior to labeled MGSA
2xIUR addition. In
addition, competitor DNAs including wild-type and mutant MGSA
2xIUR, LBP, and NF-IL6 oligonucleotides were incubated with nuclear
extracts prior to probe addition (lanes 6-12). The
resulting protein-DNA complexes were analyzed on 0.5
TBE
polyacrylamide gels; the specific IUR bound factors (IUR-F) and nonspecific complexes (ns) are
designated. The arrow indicates the nonspecific complex
present in cytoplasmic extracts.
The point mutations that did
not compete for the bound IUR complexes were identical to those that
demonstrated a loss of basal and cytokine-induced MGSA/GRO
promoter activity when placed in the MGSA
350/CAT constructs (Fig. 3). Although the IUR sequences were similar to several
consensus DNA binding elements including C/EBP and LBP sites, antisera
to these transcription factors had no effect on the bound complexes (Fig. 6B, lanes 1-5). In addition, the
consensus elements for LBP and NF-IL6 did not remove the bound
complexes (Fig. 6B, lanes 11 and 12).
Further analysis of the IUR sequence was performed by creating
additional point mutations in the MGSA/GRO -93 to -77
region present in the oligonucleotide MGSA
2xIUR. Several mutant
IUR oligonucleotides (MT 1, MT 2, MT 3) (lanes 7-9) did
not compete, while the WT and MT 4 effectively removed the bound
complexes (lanes 6 and 10). Collectively, these data
indicated that the IUR included the sequence TCGAT located at position
-97 to -93.
Figure 7:
RelA transactivates through both the
NF-B and IUR in MGSA/GRO
. RPE cells were co-transfected with
10 µg of WT, mutant IUR (mIUR), or mutant NF-
B (m
B) or double mutant (mIUR+m
B)
MGSA
350/CAT, 5 µg of cytomegalovirus-driven RelA expression
vector, and 2 µg of pCMVhGH. The total amount of DNA was held
constant by the addition of the parental pCMV4 expression vector.
Transfection efficiencies were normalized by immunodetection of
secreted growth hormone. Cells were collected 48 h post-transfection
with two medium changes. Results are expressed as -fold induction
relative to the MGSA
350/CAT alone. -Fold induction, standard
deviation of error for four separate transfections were
MGSA
350/CAT alone (1.00) RelA
(11.10 ± 0.96);
mutant IUR MGSA
350/CAT alone (0.62 ± 0.43) RelA
(8.63 ± 4.85); mutant NF-
B MGSA
350/CAT alone
(0.46 ± 0.15) RelA
(3.49 ± 1.23); and double
mutant MGSA
350/CAT alone (0.20 ± 0.19) RelA (0.21 ±
0.17). Using a nonparametric rank sum two-sided test, the mean of the
RelA transfectants (indicated by the asterisk) were
significantly different from control with an
value of
0.014.
To
address whether RelA directly bound to the MGSA/GRO IUR, RelA
produced by transfected Jurkat T-cells was used in gel mobility shift
analysis. RelA specifically recognized the MGSA/GRO
and IL-8
NF-
B elements, although not the mutated MGSA/GRO
NF-
B
element. Furthermore, RelA does not bind to the MGSA/GRO
IUR nor
enhance the binding ability of the IUR bound complexes present in
nuclear extracts (data not shown). These data suggest that RelA
indirectly effects transactivation through the MGSA/GRO
IUR.
Figure 8:
Basal
promoter activity through MGSA/GRO IUR. RPE cells were transfected
with 10 µg of the indicated TATA/CAT construct. Transfection
efficiencies were normalized by immunodetection of secreted growth
hormone. At 48 h after transfection, whole cell extracts were collected
as described under ``Materials and Methods.'' Results are
relative to CAT activity for parental TATA/CAT, to which a value of 1.0
was assigned. Values with standard deviation of error from duplicates
of three separate experiments were TATA/CAT (1.0),
MGSA
-97/-62/TATA (6.25 ± 2.30), MGSA
2xIUR/TATA (9.40 ± 3.50), and mutant IUR MGSA
2xm.IUR/TATA
(0.50 ± 0.42).
Figure 9:
UV cross-linking analysis of IUR bound
complexes. Nuclear extracts (5 µg) from IL-1 stimulated RPE cells
were incubated in 20 µl of total reaction volume with radiolabeled
MGSA 2xIUR oligonucleotide in the presence or absence of WT or
mutant (MT) MGSA
2xIUR for 20 min at room temperature. The binding
reaction was either left at room temperature (0`) or exposed to UV
radiation for 15 min (15`). A, half of the binding reaction
(10 µl) was separated on a 6% 0.5
TBE native polyacrylamide
gel. The specific IUR (IUR-F) and nonspecific (ns) complexes are indicated. B, the remaining
binding reaction volume was separated on a 9% SDS-polyacrylamide gel.
Molecular weight standards are indicated as are two specific complexes
(approximately 68 and 50 kDa) cross-linked to the labeled MGSA
2xIUR oligonucleotide (designated I and II).
Expression of the closely related chemokine gene MGSA/GRO in
several cell types is similar to the IL-8 gene. Northern analyses
demonstrates that the Hs294T melanoma cells have a high constitutive
level of both IL-8 and MGSA/GRO that is further induced by IL-1. Normal
RPE cells have a very low constitutive level of MGSA/GRO and IL-8
expression; however, IL-1 stimulation markedly increases MGSA/GRO and
IL-8 within 2 h. Previous data have demonstrated that the NF-B
element is essential for both IL-8 and MGSA/GRO promoter
activity(15, 35, 36, 37) .
Furthermore, IL-8 gene regulation requires the C/EBP site adjacent to
the NF-
B site for complete cytokine
induction(36, 38, 40) . Similar to IL-8, the
MGSA/GRO
promoter contains a region adjacent to the NF-
B
element that is necessary for basal activity in both RPE and the Hs294T
melanoma cells. Loss of either the IUR or the NF-
B element
eliminates most of the endogenous promoter activity. Cytokine-induced
MGSA/GRO
promoter activity in RPE cells also requires both the
NF-
B element and the IUR. However, we demonstrate here that the
regions adjacent to NF-
B for the IL-8 and MGSA/GRO
chemokines
differ markedly in their capacity to bind transactivating factors. For
IL-8, the C/EBP-like consensus sequence binds several C/EBP proteins,
although with weaker affinity than established C/EBP sites. In
contrast, although there is sequence similarity to a C/EBP enhancer,
C/EBP proteins do not bind the IUR in the MGSA/GRO
promoter.
Moreover, several of the essential nucleotides in the IUR are located
upstream from the C/EBP-like region.
Gel shift analyses demonstrate
that it is difficult to detect a factor specific for the IUR using
nuclear extraction procedures that give optimal NF-B binding. This
may in part be due to the observations that the IUR complexes require a
more vigorous extraction procedure, which negatively affects NF-
B
binding, and that the IUR complexes are labile after nuclear extract
collection (data not shown). Alternatively, the failure to detect
specific IUR complexes from nuclear extracts may be due to an unstable
interaction with the labeled promoter regions. Specific MGSA/GRO
IUR-bound complexes are readily observed in vitro from both
RPE and Hs294T nuclear extracts when two copies of the IUR are present,
suggesting an increased affinity with duplicate copies. The
observations that a single IUR element can compete for complex binding
yet not detectably bind nuclear factors in gel shift assays suggest
that the protein complexes bound to a single IUR may be unstable during
electrophoresis.
The IUR bound complexes are effectively competed by
oligonucleotides containing a single copy of the IUR without the
NF-B element, suggesting the IUR complexes bind the DNA
independent of the NF-
B element. Furthermore, a series of point
mutations narrows the IUR to a span of nucleotides containing the
sequence TCGAT. This sequence does not have any similarities to known
transcription factor sequences, suggesting that the factors that bind
to the IUR are novel. UV cross-linking indicates the presence of at
least two proteins of approximately 22 and 40 kDa bound to the
MGSA/GRO
IUR. These IUR-bound complexes are constitutively
present, and IL-1 induction does not further increase the binding
activity.
In addition to the NF-B element, RelA is able to
transactivate through the IUR region within the MGSA/GRO
promoter.
These observations are similar to those observed with IL-6 and IL-8
gene regulation in that both the adjacent C/EBP-like and NF-
B
elements are essential for cytokine induction and RelA
transactivation(40) . However, our results differ in that RelA
alone is able to transactivate the MGSA/GRO
promoter and does not
require an additional factor to be co-transfected.
The RelA
induction through the IUR alone does not dictate that RelA
cross-couples with the IUR-bound complexes as demonstrated for the IL-8
gene. RelA antisera has no effect on the IUR-bound complexes nor does
RelA enhance the binding of the IUR complexes; therefore, a direct
interaction between NF-B and IUR-bound complexes is not currently
supported by our data. The IUR complexes do significantly contribute to
MGSA/GRO
basal promoter activity in that loss of the IUR complexes
bound to the endogenous MGSA/GRO
promoter, or to a minimal
promoter containing only the IUR sequences, dramatically decrease the
amount of activity obtained from the native promoter. RelA may act
indirectly to either induce expression of the IUR binding proteins or
stabilize their interaction with the basal transcription machinery.
In summary, our results indicate that, as with other genes encoding
proteins involved in the inflammatory response including
IL-6(40) , IL-8(36, 38, 39) , serum
amyloid A genes(52, 60) , and angiotensinogen (61) , MGSA/GRO transcriptional regulation requires
multiple factors recognizing the NF-
B and adjacent DNA binding
elements. However, the IUR adjacent to the NF-
B element in the
MGSA/GRO
promoter appears to be unique. In addition, RelA has a
dual role in MGSA/GRO
activation in that it is able to
transactivate through both the NF-
B element and the adjacent IUR.
Since NF-
B is ubiquitous and a multitude of genes contain
NF-
B elements, the regulation by RelA through a separate enhancer
region may allow a tighter and more specific level of gene regulation.
Future studies are needed to determine the mechanism by which this
region interacts with the adjacent NF-
B element to regulate
MGSA/GRO transcription in normal and transformed cells.