©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Constitutive and Cytokine-induced Expression of the Melanoma Growth Stimulatory Activity/GRO Gene Requires Both NF-B and Novel Constitutive Factors (*)

(Received for publication, September 25, 1995)

Lauren D. Wood (1) Ann Richmond (1) (2) (3)(§)

From the  (1)Department of Cell Biology and the (2)Department of Medicine, Division of Dermatology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2175 and the (3)Veterans Affairs Medical Center, Division of Dermatology, Nashville, Tennessee 37212-2637

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-kappaB 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/GROalpha in normal retinal pigment epithelial and the Hs294T melanoma cells is partially regulated through the NF-kappaB element, which binds both NF-kappaB p50 and RelA (NF-kappaB p65) homodimers and heterodimers. Mutational analysis of the MGSA/GROalpha promoter reveals that, in addition to the NF-kappaB 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-kappaB element or the IUR, suggesting a putative interaction between NF-kappaB and this novel complex.


INTRODUCTION

Chemokines play a major role in mediation of inflammation and wound healing. Melanoma growth stimulatory activity (MGSA(^1)/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/GROalpha, -beta, and -)(28, 29, 30, 31) . The MGSA/GROalpha form was purified from culture medium conditioned by Hs294T human melanoma cells(16, 32, 33) , and overexpression of MGSA/GROalpha 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/GROalpha gene that cannot be significantly induced by the cytokines IL-1 and tumor necrosis factor alpha. In contrast, IL-1 markedly increases MGSA/GROalpha gene transcription in normal retinal pigment epithelial (RPE) cells(35) .

The human IL-8 and MGSA/GRO genes contain an NF-kappaB 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-kappaB element(36) . The C/EBP and NF-kappaB 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-kappaB element, the immediate upstream region (IUR) is necessary for basal and cytokine induced expression of MGSA/GROalpha in RPE cells. Likewise, basal MGSA/GROalpha promoter activity within the Hs294T melanoma cells also requires this region. However, unlike the IL-8 promoter, neither C/EBPalpha nor C/EBPbeta recognize the similar region in the MGSA/GROalpha promoter. Furthermore, RelA (NF-kappaB p65) transactivates MGSA/GROalpha transcription either directly through the NF-kappaB 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/GROalpha gene involves multiple factors that recognize the NF-kappaB and immediate surrounding regions.


MATERIALS AND METHODS

Northern Blot Analysis

Hs294T and RPE cells were cultured as described by Shattuck et al.(35) . Total RNA was purified as described previously(35) . Random primed 700-base pair EcoRI fragment of MGSA/GROalpha cDNA (16) and a 400-base pair EcoRI fragment of IL-8 cDNA (41) were used as probes. Hybridization of cyclophilin (1B15) was used as a standard for quantitation.

CAT Reporter Gene Plasmid Construction

MGSAalpha350/CAT and mutant NF-kappaB MGSAalpha350/CAT constructs were described earlier(35) . Mutation of the IUR within the context of the MGSAalpha350/CAT constructs was achieved by recombinant polymerase chain reaction using polymerase chain reaction primers containing the mutations underlined 5`-GGGATCGACCTGGGTCTCCG-3`. Site-directed mutagenesis was utilized to generate an additional mutation within the IUR element (IUR-B) (see Table 1) using the Altered Sites mutagenesis system (Promega). The expected point mutations were confirmed by restriction enzyme digestion and sequencing. MGSAalpha(-97/-62)TATA/CAT, MGSAalpha 2xIUR, and MGSAalpha m.2xIUR were generated by ligating the respective annealed oligonucleotides (see Table 1) into the cloning region in the parental TATA/CAT vector (39) .



Expression Vector Constructs and Recombinant Protein

The RelA expression vector was as described previously (42) and was the gift of Warner Greene (University of California, San Francisco). NF-IL6 cDNA was as described previously (43) and was the gift of Tadamitsu Kishimoto (Osaka University, Osaka, Japan). Recombinant C/EBPbeta and NF-IL6 were produced utilizing pRSET vectors as described previously (44) and were the generous gifts of Linda Sealy (Vanderbilt University).

Transfection and CAT Reporter Assay

Either RPE or Hs294T cells were co-transfected with 10 µg of the indicated MGSA/CAT fusion genes (for point mutations see Table 1) and 2 µg of pCMVhGH (obtained from Dr. Lynn Matrisian, Vanderbilt University), which allowed for normalization of transfection efficiency by measuring growth hormone secretion utilizing the immunoassay (Nichols Institute). Transfections were performed by the calcium phosphate coprecipitation method(45) . CAT enzymatic activity was assayed as described previously(46) . The percent [^14C]chloramphenicol converted to acetylated forms was determined by PhosphorImage analysis (Molecular Dynamics).

Radiolabeled and Competitor DNA

Oligonucleotides were synthesized on a Milligen 7500 DNA synthesizer (Diabetes Research DNA Core, Vanderbilt University). Equal amounts of each oligonucleotide and its complement were annealed in STE (10 mM Tris, pH 7.8, 1 mM EDTA, pH 8.0, 200 mM NaCl) by boiling the oligonucleotides in a water bath that was slowly cooled to room temperature (approximately 4 h). The oligonucleotides (coding strand) are shown in Table 1. Probes for gel mobility shift analyses were prepared by radiolabeling 100 ng of annealed oligonucleotides with T4 polynucleotide kinase.

Nuclear Extracts and DNA Binding Assay

Nuclear extracts were prepared from Hs294T and RPE cells as described previously (35) with the exception that cell lysis was performed by vortexing vigorously in the presence of buffer A with 1% Nonidet P-40. DNA binding reactions with recombinant C/EBPbeta and NF-IL6 proteins were performed by combining the indicated amounts of recombinant proteins in 20 µl of 19 mM Hepes, pH 8.0, 1 mM Tris, pH 8.0, 50 mM NaCl, 10 mM KCl, 0.18 mM EDTA, pH 8.0, 1 mM spermidine, 10% glycerol, 0.5% Triton X-100, 0.1% Nonidet P-40, 0.4 µg of BSA, 10.2 mM dithiothreitol, and 1 µg poly(dI-dC)bulletpoly(dI-dC) for 20 min at 37 °C prior to probe addition (40,000 counts/reaction) for 20 min at room temperature. The binding reaction was then analyzed by electrophoresis in a nondenaturing 6% polyacrylamide gel in 0.25 times TBE (22 mM Tris, pH 8.0, 22 mM boric acid, 0.5 mM EDTA, pH 8.0). DNA binding reactions with Hs294T and RPE nuclear extracts were performed by incubating 5-µg nuclear extracts in 10 mM Tris, pH 8.0, 50 mM NaCl, 5% glycerol, and 1 µg of poly(dI-dC)bulletpoly(dI-dC) for 15 min prior to probe addition (20,000 counts) for 20 min at room temperature. The resulting protein-DNA complexes were separated on 6% polyacrylamide gel in 0.5 times TBE (45 mM Tris, pH 8.0, 45 mM boric acid, 1 mM EDTA, pH 8.0). Jurkat T lymphocytes were transfected with a RelA expression vector or parental pCMV4 vector alone by electroporation as described previously(47) . Whole cell extracts were prepared from Jurkat T-cell transfectants by high salt extraction as described previously(47) . Gel shift assays were performed as above with 10 µg of Jurkat extract.

UV Cross-linking

Nuclear extracts (5 µg) incubated with the labeled MGSAalpha 2xIUR oligonucleotide (20 µl total reaction volume) were exposed to short wave UV radiation for 15 min (Stratalinker). Half of the reaction (10 µl) was separated on a 6% nondenaturing gel as described for the DNA binding assay. The remaining 10 µl was heated (95 °C) for 5 min in SDS loading buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 1% beta-mercaptoethanol, 0.1% bromphenol blue). Labeled proteins were then separated by electrophoresis on a 9% SDS-polyacrylamide gel and compared with molecular weight standards.

Antibodies

Antibodies to NF-kappaB p50, RelA and preimmune antisera (42, 48) were the kind gift of Warner Greene. In addition, antisera to RelA, NF-kappaB p50, NF-kappaB p52, and C/EBPbeta were obtained from Santa Cruz. Additional antibodies directed against C/EBPalpha, C/EBPbeta, and C/EBP were the generous gift of Stephen McKnight and were as described previously(49) . Anti-leader binding protein (LBP) antisera was generously provided by Robert Roeder(50) .


RESULTS

Regulation of MGSA/GRO and IL-8 mRNA Synthesis by IL-1

MGSA/GRO and IL-8 mRNA levels increased rapidly in normal RPE cells stimulated with IL-1 ( Fig. 1and (35) ). This induction of MGSA/GRO was primarily due to increased transcription(35) . The Hs294T melanoma cells have a constitutive level of both MGSA/GRO and IL-8 gene expression (Fig. 1). The basal level of activity of both MGSA/GRO and IL-8 in the Hs294T cells was equal to or greater than the IL-1-induced level in the RPE cells. We had previously demonstrated by promoter deletion studies that the MGSA/GROalpha region between -100 and -43 from the transcription start site were necessary for basal expression in Hs294T melanoma cells and basal and cytokine-induced expression in RPE cells(35) . Further analysis demonstrated that the NF-kappaB element within this region was necessary for this activation.


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-kappaB and the adjacent C/EBP binding elements were required for IL-1 and tumor necrosis factor alpha 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-kappaB directly interacted with the C/EBP complexes (38, 39) . Sequence analysis between the IL-8 and MGSA/GROalpha promoter region indicated an almost identical NF-kappaB element and adjacent C/EBP-like region (Fig. 2). The MGSA/GROalpha nucleotide sequence adjacent to the NF-kappaB 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-kappaB 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/GROalpha promoter activity required both NF-kappaB- and Sp1-related complexes bound within the immediate promoter(57) .


Figure 2: Sequence comparison of MGSA/GROalpha-97/-62 and IL-8-100/-65 with several consensus transcription factor elements. Sequence comparison of MGSA/GROalpha-97/-62 and IL-8-100/-65 with several consensus transcription factor elements. The nucleotide sequences of the MGSA/GROalpha -97/-62 and IL-8 -100/-65 promoter regions containing the NF-kappaB element are shown. The shaded boxes represent the NF-kappaB element and either the NF-IL6 or the IUR in the IL-8 and MGSA/GROalpha promoter, respectively. Nucleotides that are not conserved between the indicated MGSA/GROalpha 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-kappaB p65 consensus(62) , IL-8 (36) , immunoglobulin kappa light chain enhancer (IgG kappa)(63) . The HIV long terminal repeat LBP-1 binding site sequence is included(53, 54) .



Immediate Upstream Region Is Required for MGSA/GROalpha Promoter Activity

To determine if the IUR located adjacent to the NF-kappaB element was involved in MGSA/GROalpha regulation as demonstrated for IL-8 gene regulation(36, 38, 58) , we studied the activities of MGSAalpha350/CAT constructs with point mutations in both the NF-kappaB and upstream region in transiently transfected RPE cells. Mutations in either site resulted in a substantial loss of basal CAT activity (>50%) and subsequent loss of IL-1 induction in RPE cells (Fig. 3A). Mutations made in both regions resulted in a complete loss of CAT activity comparable with the activity obtained from the parental vector pPLFCAT, indicating a complete loss of activation through the MGSA/GROalpha promoter region (Fig. 3A). Likewise, within the Hs294T melanoma cell line, mutation of either the NF-kappaB or the IUR resulted in a substantial loss in the base-line CAT activity (Fig. 3B). These results indicated that, in addition to the consensus NF-kappaB element, the IUR element plays an important role in MGSA/GROalpha gene regulation in both RPE and Hs294T cells.


Figure 3: IUR is required for MGSA/GROalpha promoter activity in RPE and Hs294T cells. IUR is required for MGSA/GROalpha promoter activity in RPE and Hs294T cells. RPE (A) or Hs294T (B) cells were co-transfected with 10 µg of the indicated MGSAalpha350/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 MGSAalpha350/CAT alone with no treatment. -Fold induction, standard deviation of error, and number of separate transfections were MGSAalpha350/CAT unstimulated (1.00) IL-1 (5.18 ± 1.65) (n = 5), mutant IUR MGSAalpha350/CAT unstimulated (0.26 ± 0.18) IL-1 (0.46 ± 0.26) (n = 4), mutant NF-kappaB MGSAalpha350/CAT unstimulated (0.40 ± 0.28) IL-1 (0.41 ± 0.18) (n = 5), and double mutant MGSAalpha350/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 MGSAalpha350/CAT wild-type (7.22 ± 1.19), mutant IUR (0.09 ± 0.07), and mutant NF-kappaB (0.70 ± 0.28).



Characterization of the Nuclear Proteins That Bind to the MGSA/GROalpha-97/-62 Region

Gel mobility shift analyses were performed to characterize the nuclear factors that bind to the IUR and NF-kappaB region. Nuclear extracts from RPE cells either untreated or IL-1 treated were incubated with either the wild type (WT), mutant IUR (m.IUR), mutant NF-kappaB (m.kappaB) or double mutant (m.IUR+m.kappaB) MGSAalpha-97/-62 probe (Fig. 4). IL-1 induction resulted in the appearance of two shifted nuclear complexes bound to the wild-type MGSAalpha-97/-62 oligonucleotide (lanes 1 and 2). Addition of various NF-kappaB antisera indicated that the lower complex consisted of the NF-kappaB p50/RelA heterodimer, while the upper complex contained RelA (presumably homodimers) (lanes 4 and 5). Addition of preimmune, NF-kappaB p52 and C/EBPbeta antisera had no effect on the shifted complexes (lanes 3, 5, and 7). Gel mobility shift analyses with labeled mutant IUR (m.IUR) MGSAalpha-97/-62 oligonucleotide demonstrated the identical pattern of shifted complexes as wild-type oligonucleotide (lanes 8 and 9). Labeled mutant NF-kappaB (m.kappaB) or double mutant (m.IUR+m.kappaB) MGSAalpha-97/-62 oligonucleotides did not retard nuclear complexes (lanes 10-13). These data demonstrated that the nuclear complexes bound to the MGSA/GROalpha -97/-62 region are specific for the NF-kappaB element and not the IUR.


Figure 4: Characterization of nuclear proteins that bind to the MGSA/GROalpha -97/-62 region. Characterization of nuclear proteins that bind to the MGSA/GROalpha -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-kappaB (m.kappaB) or double mutant (m.IUR+m.kappaB) MGSAalpha-97/-62 oligonucleotides. Preimmune (PI) antisera or antisera to NF-kappaB p50, NF-kappaB p52, RelA, or C/EBPbeta were incubated with extracts prior to addition of labeled probe. The resulting protein-DNA complexes were separated on 0.5 times TBE polyacrylamide gels. The NF-kappaB p50/RelA heterodimer and RelA complexes are indicated.



Characterization of MGSA/GROalpha C/EBP-like Sequence as a Potential C/EBP Binding Element

Several groups have demonstrated cooperative binding of C/EBP family members with RelA to the IL-8 C/EBP enhancer region(38, 39) . However, IL-8 has a weak binding affinity for C/EBP proteins. Using the IL-8 or MGSA/GROalpha C/EBP-like regions as probes, we have not been able to identify a C/EBP-related complex from RPE or Hs294T nuclear extracts, although C/EBP proteins were present in these extracts based on immunoblot and gel shift analysis with the NF-IL6 consensus oligonucleotide as a probe (data not shown). Purified C/EBPbeta or NF-IL6 did not bind to the C/EBP-like region in the MGSA/GROalpha promoter, although it did bind weakly to the IL-8 C/EBP enhancer region (data not shown). We addressed the possibility that the affinity of C/EBPbeta for the MGSA/GROalpha enhancer region was too weak to detect an interaction after electrophoresis during gel shift analysis. A converse approach was to analyze the ability of the IL-8 and MGSA/GROalpha regions to compete for C/EBPbeta protein bound to the labeled NF-IL6 consensus element (Fig. 5). Unlabeled NF-IL6 oligonucleotide at 5-fold excess to radiolabeled NF-IL6 probe reduced C/EBPbeta binding by >90% (Fig. 5). With 50-fold excess NF-IL6 oligonucleotide, the C/EBPbeta complex was completely removed. Unlabeled IL-8-101/-63 oligonucleotide reduced C/EBPbeta binding to the NF-IL6 oligonucleotide by >70% at 200-fold excess, while addition of higher concentrations of this oligonucleotide completely removed C/EBPbeta binding. In contrast, addition of unlabeled MGSAalpha-97/-62 did not reduce C/EBPbeta binding even at 2000-fold excess (Fig. 5C). Our data agreed with earlier studies in which purified C/EBPbeta bound weakly to the IL-8 enhancer region as compared to the NF-IL-6 element (38, 39) . Moreover, these results indicated that although there were nucleotide similarities between the MGSAalpha-97/-62 region and previously identified C/EBP consensus elements, the MGSA/GROalpha region adjacent to the NF-kappaB element did not bind C/EBPbeta proteins.


Figure 5: Characterization of C/EBP-like elements in the MGSA/GROalpha and IL-8 promoter. Characterization of C/EBP-like elements in the MGSA/GROalpha and IL-8 promoter. Recombinant C/EBPbeta 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, MGSAalpha-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 times TBE polyacrylamide gels. Quantitation of bound protein-DNA complexes was done using the software program IQ3.29 for the PhosphorImager (Molecular Dynamics).



Identification of IUR-bound Complexes by Gel Mobility Shift Analysis

Since the majority of the nuclear complexes that recognized the MGSA/GROalpha -97/-62 region were NF-kappaB-related, a 42-base pair probe was prepared that contained two copies of the IUR without the NF-kappaB element present (designated MGSAalpha 2xIUR). When this probe was used in gel mobility shift assays with nuclear extracts from either RPE or Hs294T cells, two complexes bound (IUR-F) (Fig. 6, A and B). Cytoplasmic extracts demonstrated the presence of a slower migrating complex that appeared to be nonspecific in that all MGSA/GROalpha oligonucleotides tested including wild-type and mutants removed this complex in competition analysis. Unlabeled competitor DNAs were used to test the specificity of the nuclear IUR-F complexes bound to the labeled MGSAalpha 2xIUR. An oligonucleotide from the IL-8 promoter containing the C/EBP and NF-kappaB elements did not compete (Fig. 6A, lane 6), while wild-type MGSAalpha-97/-62 efficiently removed the IUR-bound complexes (lane 7). Point mutations in three nucleotides in the IUR (m.IUR) resulted in loss of competition (lanes 8 and 10), while other point mutations in the IUR (m.IUR-B, m.IUR-C) and the NF-kappaB element (m.kappaB) competed as wild-type (lanes 9, 11-12). A shorter oligonucleotide (WT MGSAalpha-97/-78) that does not have the NF-kappaB element present also effectively competed the bound complexes (lane 13), while point mutations in the IUR region did not (lane 14), suggesting the complexes specifically recognized the IUR independent of the NF-kappaB element.


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 MGSAalpha 2xIUR oligonucleotide. Unlabeled competitor probes (50-fold excess) included IL-8 (lane 6), wild-type, and mutant MGSAalpha-97/-62 and MGSAalpha-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/EBPbeta, and LBP (lanes 3-5) 20 min prior to labeled MGSAalpha 2xIUR addition. In addition, competitor DNAs including wild-type and mutant MGSAalpha 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 times 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/GROalpha promoter activity when placed in the MGSAalpha350/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/GROalpha -93 to -77 region present in the oligonucleotide MGSAalpha 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.

Activation of MGSA/GROalpha Promoter by RelA

Previous work has demonstrated a cross-coupling of NF-kappaB and C/EBP family members(39, 59) . In particular, these investigators demonstrated that regulation of the chemokine IL-8 gene expression relied on the ratio of NF-kappaB and C/EBP complexes in that the C/EBPbeta protein had an inhibitory effect through the adjacent NF-kappaB element, while RelA enhanced transactivation through the C/EBP element(39) . We were interested in determining if RelA regulated MGSA/GROalpha gene expression similarly through the NF-kappaB and adjacent regions in a manner similar to that demonstrated for the IL-8 gene(38, 39, 40) . RPE cells were transiently co-transfected with a RelA expression vector, and the MGSAalpha350/CAT reporter constructs with point mutations in the IUR and NF-kappaB element. Overexpression of RelA increased activity through both the MGSAalpha350/CAT and the mutant IUR MGSAalpha350/CAT (Fig. 7). Moreover, RelA also increased transactivation through the mutant NF-kappaB MGSAalpha350/CAT. The transactivation seen with the mutant NF-kappaB construct was less than that observed with the mutant IUR construct. Furthermore, a second set of mutations in the NF-kappaB element (GAAAATTTGGC) within the context of MGSAalpha350/CAT indicated that RelA still significantly increased promoter activity (data not shown). RelA expression did not transactivate through either the double mutant MGSAalpha350/CAT or parental pPLFCAT vectors, suggesting that the RelA transactivation observed with the mutant NF-kappaB element construct was through the adjacent IUR region (Fig. 7).


Figure 7: RelA transactivates through both the NF-kappaB and IUR in MGSA/GROalpha. RPE cells were co-transfected with 10 µg of WT, mutant IUR (mIUR), or mutant NF-kappaB (mkappaB) or double mutant (mIUR+mkappaB) MGSAalpha350/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 MGSAalpha350/CAT alone. -Fold induction, standard deviation of error for four separate transfections were MGSAalpha350/CAT alone (1.00) RelA (11.10 ± 0.96); mutant IUR MGSAalpha350/CAT alone (0.62 ± 0.43) RelA (8.63 ± 4.85); mutant NF-kappaB MGSAalpha350/CAT alone (0.46 ± 0.15) RelA (3.49 ± 1.23); and double mutant MGSAalpha350/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 alpha value of 0.014.



To address whether RelA directly bound to the MGSA/GROalpha IUR, RelA produced by transfected Jurkat T-cells was used in gel mobility shift analysis. RelA specifically recognized the MGSA/GROalpha and IL-8 NF-kappaB elements, although not the mutated MGSA/GROalpha NF-kappaB element. Furthermore, RelA does not bind to the MGSA/GROalpha 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/GROalpha IUR.

IUR Contributes to Basal MGSA/GROalpha Expression

Gel shift analysis indicated that several key nucleotides in the IUR were essential for recognition by the constitutive complexes in both RPE and Hs294T melanoma cells. Furthermore, these point mutations strongly effected basal and cytokine-induced MGSAalpha350/CAT activity, suggesting that the bound IUR complexes, in addition to the NF-kappaB complexes, contributed to the transcriptional regulation of MGSA/GROalpha. Minimal promoter contructs containing a single copy of the IUR plus the NF-kappaB element (MGSAalpha(-97/-62)TATA) or two copies of the wild type (MGSAalpha 2xIUR/TATA) or mutated IUR (MGSAalpha m.2xIUR/TATA) were generated. Transient transfections in RPE cells demonstrated that both the wild-type MGSAalpha(-97/-62) and MGSAalpha 2xIUR/TATA constructs had a higher level of basal promoter activity as compared with the parental TATA/CAT (Fig. 8). Furthermore, point mutations in the IUR, which resulted in loss of the constitutively bound IUR complexes effectively eliminated all basal promoter activity (Fig. 8). Together with the endogenous MGSA/GROalpha promoter analyses, these data indicated that the IUR-bound complexes significantly contributed to MGSA/GROalpha basal promoter activity.


Figure 8: Basal promoter activity through MGSA/GROalpha 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), MGSAalpha-97/-62/TATA (6.25 ± 2.30), MGSAalpha 2xIUR/TATA (9.40 ± 3.50), and mutant IUR MGSAalpha 2xm.IUR/TATA (0.50 ± 0.42).



Identification of IUR-bound Complexes by UV Cross-linking

To further characterize the complexes bound to the IUR, UV cross-linking studies were performed. Nuclear proteins bound to the labeled MGSAalpha 2xIUR in the gel shift reactions were either left untreated or exposed to short-wave UV irradiation for 15 min. Present in the reaction was 50-fold excess of either the wild-type or mutant MGSAalpha 2xIUR oligonucleotides. Half of the reaction was separated on a native 6% polyacrylamide gel (Fig. 9A). The wild-type MGSAalpha 2xIUR oligonucleotide (WT) effectively competed the upper bound complexes, while the mutant MGSAalpha 2xIUR oligonucleotide (MT) did not. UV radiation did not affect the bound complexes or the competition analysis (Fig. 9A, compare lanes 1-3 to lanes 4-6). The remaining half of the binding reaction was separated on a 9% SDS-polyacrylamide gel (Fig. 9B). Two complexes were cross-linked to the labeled probe and appeared to be specific in that the wild-type oligonucleotide significantly lessened their presence, while mutant oligonucleotide did not (Fig. 9B, lanes 13). These complexes were not observed when the binding reactions were not exposed to UV radiation (Fig. 9B, lanes 4-6). In these cross-linking studies, there were not specific complexes detected below the 40-kDa marker on the SDS-polyacrylamide gel (data not shown). The cross-linked complexes observed were approximately 68 and 50 kDa; however, the 42-base pair oligonucleotide radiolabeled probe present in the cross-linked complexes contributed approximately 28 kDa. Therefore, the two IUR bound complexes are within the 22-40 kDa range. These data indicated that multiple DNA binding proteins bind to the MGSA/GROalpha IUR.


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 MGSAalpha 2xIUR oligonucleotide in the presence or absence of WT or mutant (MT) MGSAalpha 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 times 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 MGSAalpha 2xIUR oligonucleotide (designated I and II).




DISCUSSION

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-kappaB 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-kappaB site for complete cytokine induction(36, 38, 40) . Similar to IL-8, the MGSA/GROalpha promoter contains a region adjacent to the NF-kappaB element that is necessary for basal activity in both RPE and the Hs294T melanoma cells. Loss of either the IUR or the NF-kappaB element eliminates most of the endogenous promoter activity. Cytokine-induced MGSA/GROalpha promoter activity in RPE cells also requires both the NF-kappaB element and the IUR. However, we demonstrate here that the regions adjacent to NF-kappaB for the IL-8 and MGSA/GROalpha 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/GROalpha 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-kappaB binding. This may in part be due to the observations that the IUR complexes require a more vigorous extraction procedure, which negatively affects NF-kappaB 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/GROalpha 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-kappaB element, suggesting the IUR complexes bind the DNA independent of the NF-kappaB 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/GROalpha IUR. These IUR-bound complexes are constitutively present, and IL-1 induction does not further increase the binding activity.

In addition to the NF-kappaB element, RelA is able to transactivate through the IUR region within the MGSA/GROalpha 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-kappaB elements are essential for cytokine induction and RelA transactivation(40) . However, our results differ in that RelA alone is able to transactivate the MGSA/GROalpha 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-kappaB and IUR-bound complexes is not currently supported by our data. The IUR complexes do significantly contribute to MGSA/GROalpha basal promoter activity in that loss of the IUR complexes bound to the endogenous MGSA/GROalpha 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/GROalpha transcriptional regulation requires multiple factors recognizing the NF-kappaB and adjacent DNA binding elements. However, the IUR adjacent to the NF-kappaB element in the MGSA/GROalpha promoter appears to be unique. In addition, RelA has a dual role in MGSA/GROalpha activation in that it is able to transactivate through both the NF-kappaB element and the adjacent IUR. Since NF-kappaB is ubiquitous and a multitude of genes contain NF-kappaB 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-kappaB element to regulate MGSA/GRO transcription in normal and transformed cells.


FOOTNOTES

*
This research was supported in part by National Institutes of Health Grants CA56704 and 5P30 AR41943, American Cancer Society Grant BE146, a Department of Veterans Affairs Merit and Associate Career Scientist award (to A. R.); and the Vanderbilt University Graduate Fellowship award (to L. D. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 615-343-7777; Fax: 615-343-4539.

(^1)
The abbreviations used are: MGSA, melanoma growth stimulatory activity; GRO, growth regulated; IL, interleukin; DARC, Duffy antigen receptor for chemokines; RPE, retinal pigment epithelial; IUR, immediate upstream region; LBP, leader binding protein; HIV-1, human immunodeficiency virus type I; CAT, chloramphenicol acetyltransferase; WT, wild-type.


ACKNOWLEDGEMENTS

We thank Drs. Steven McKnight, Linda Sealy, Glenn Jaffe, Bernd Stein, Albert Baldwin, Warner Greene, Robert Roeder, Kouji Matsushima and Tadamitsu Kishimoto for valuable reagents. We also thank Drs. Larry Kerr, Linda Sealy and Rebecca Shattuck for helpful discussions.


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