(Received for publication, August 16, 1995; and in revised form, September 19, 1995)
From the
We have recently described the isolation of a novel protein, MIA, which is secreted from malignant melanoma cells and elicits growth inhibition on melanoma cells in vitro (Blesch, A., Bosserhoff, A. K., Apfel, R., Behl, C., Hessdörfer, B., Schmitt, A., Jachimczak, P., Lottspeich, F., Schlingensiepen, H., Buettner, R., and Bogdahn, U.(1994) Cancer Res. 54, 5695-5701). Here, we report the structure of the human MIA gene locus, describe its expression pattern in melanocytic tumors in vivo, and provide an initial characterization of the MIA promoter. The MIA gene is encoded by four exons, and the mRNA initiation site was identified 70 base pairs upstream from the translation start codon. MIA mRNA expression in vivo correlated with progressive malignancy of melanocytic lesions and was inducible in other cells by phorbol esters. To investigate mechanisms mediating this melanoma-associated expression pattern, we analyzed the promoter activity of the 1.3-kilobase genomic sequences located 5`-upstream of the MIA gene. The MIA promoter conferred high levels of gene activation specifically in human and murine melanoma cells, and its activity was further enhanced by treatment with phorbol esters. Site-directed mutation of an NF-kB site within the MIA promoter did reduce the basal promoter activity in melanoma cells but did not change significantly enhancement by phorbol esters.
Growth and expansion of tumor cells including malignant
melanomas are modulated by a complex network of growth factors, which
regulate proliferation and cell-matrix interaction through a variety of
different signal transduction pathways. Therefore, the net growth or
regression of melanomas in vivo reflects integration of many
different stimulatory or inhibitory factors produced both by the tumors
cells and their environment. Well studied examples of growth regulatory
proteins in melanoma cells include members of the tumor growth
factor- and platelet-derived growth factor families, transferrin,
basic fibroblast growth factor, epidermal growth factor, and tumor
growth factor-
(Herlyn and Malkowicz, 1991; Halaban et
al., 1991; Rodeck et al., 1991; Shih and Herlyn, 1993).
We have recently isolated and cloned a novel protein that is secreted by malignant melanoma cell lines and exerts autologous growth inhibitory effects on melanoma cells in vitro (Bogdahn et al., 1989; Apfel et al., 1993; Blesch et al., 1994). Due to the growth inhibitory effect, which allowed purification by means of a bioassay, this protein was designated MIA (melanoma inhibitory activity). Isolation of fully encoding human and murine MIA cDNA clones revealed that MIA is translated as a 131-amino acid precursor protein and secreted into the tissue culture supernatant of melanoma cells after cleavage of a 24-amino acid signal peptide. MIA appears to constitute a unique protein since no significant sequence homology to any other known protein was detected.
Initial characterization of MIA expression by Northern blot analyses indicated that MIA is expressed in all melanoma cell lines that we tested and infrequently in glioma cell lines but not in fibroblast or epithelial cell lines. This interesting melanoma-associated expression pattern prompted us to examine in more detail skin biopsies along with benign and malignant melanocytic tumors in vivo for expression of MIA mRNA. We further aimed to isolate the entire genomic locus of the human MIA gene, to determine its exon-intron organization, and to provide an initial characterization of the melanoma cell type-specific function of the MIA gene promoter.
Figure 1: Exon-intron structure of the human MIA gene and relative position of all four exons. Displayed on top is a chart of the MIA protein indicating the secretion signal of the prepeptide (amino acids 1-25) and the last amino acid of all four exons (amino acids 42, 88, 125, and 131, respectively). The center chart shows two adjacent genomic XbaI fragments (UB1-1 and UB1-3) and the relative location of the MIA exons. Sequences of the exon-intron junctions are shown at the bottom. Capital letters indicate coding nucleic acid residues.
RACE-PCR ()was performed using the AmpliFinder RACE kit
(Clontech) precisely as described previously (Bauer et al.,
1994). Briefly, an antisense primer (5`-CAGCCATGGAGATAGGGT-3`) matching
residues +57 to +75 of the MIA cDNA was used for reverse
transcription of 2 µg of poly(A)
-selected RNA
isolated from Mel Im melanoma cells. After hydrolysis of the template
mRNA, an anchor primer was ligated to the 3`-end of the cDNA, and then
a PCR reaction was performed using the anchor primer and the same
oligonucleotide that was used for the primer extension analysis as a
nested MIA-specific primer.
The genomic region 5`
adjacent to the MIA gene from residue -1361 to -1
was amplified by PCR, inserted into the promoterless chloramphenicol
acetyltransferase plasmid pBLCAT3 (Luckow and
Schütz, 1987), resequenced, and then a series of
5`-deleted constructs was generated using nested deletion (Henikoff,
1984). 2 10
cells were seeded into 90-mm dishes and
transiently transfected with 5 µg of plasmid DNA using DOTAP
transfection reagent (Boehringer) the following day. To normalize
transfection efficiency, 2.5 µg of an LTR-lacZ plasmid was
cotransfected and an enzyme-linked immunosorbent assay (Boehringer) was
used to quantify CAT activities.
Figure 2:
Determination of the MIA mRNA initiation
site. A, primer extension assay. A 25-mer synthetic
oligonucleotide was phospholabeled and used to extend the N-terminal
end of the MIA mRNA. Extended products were size-fractionated on a 5%
urea/polyacrylamide gel next to a sequencing reaction as a size marker.
The largest extended product is 54 bp in length matching nucleic acid
residue -70 relative to the ATG protein start codon. B,
direct cloning of the N-terminal MIA cDNA end by RACE-PCR. First strand
cDNA was synthesized from Mel Im poly(A) RNA using an
antisense primer in the first exon. An anchor primer was ligated to the
3`-cDNA end, and the resulting template was amplified by PCR using the
anchor primer and a nested MIA primer. Shown is an ethidium-stained
agarose gel of the PCR product next to a molecular size standard. C, graphic summary indicating relative location of RACE-PCR
primers and the size of the expected PCR
product.
As shown in Fig. 3A, specific MIA RT-PCR products were readily
amplified from both melanoma cell lines but not from two different
benign melanocyte cultures. To control the specificity of these
reactions, we subcloned the PCR products into a plasmid vector and
confirmed by sequencing that they represented MIA cDNA fragments (data
not shown). In parallel -actin mRNA was amplified to verify equal
amounts and integrity of different RNA preparations.
Figure 3:
A,
amplification of MIA cDNA by RT-PCR in melanocytes and melanoma cell
lines Mel Im and HTZ-19 (left). Control PCRs were performed on
-actin mRNA in parallel (right). B,
amplification of MIA cDNA in various cell lines indicated on top.
We then used the same PCR conditions to perform PCR reactions on RNA samples from a series of different cell lines. As summarized in Table 1and in good agreement with previously performed Northern blot analyses (Blesch et al., 1994), we detected high levels of MIA mRNA expression in every melanoma cell line we tested, including B16 murine melanoma cells. In contrast, we did not detect any significant expression in other skin-derived cells including normal fibroblasts and HaCaT keratinocytes (Fig. 3B). Also, other epithelial cell lines such as COS, HeLa, and HepG2 cells, DU 145 (human prostate cancer) and J82 cells (human bladder cancer), or PA-1 teratocarcinoma cells did not express MIA. Interestingly, significant MIA expression was induced by treatment with phorbol esters in skin fibroblasts, HaCaT, COS, and HeLa cells.
These results prompted us to study the
expression pattern of MIA mRNA in normal skin and skin-derived
melanocytic tumors. As shown in Table 2and as examples in Fig. 4A, we did not detect MIA mRNA in normal skin
except for two cases, in which minute mRNA levels were amplified when
32 rather than 25 PCR cycles were performed. Low or moderate MIA mRNA
levels were detected in 8 of 15 benign melanocytic nevi, and high
levels were found in one case. In all specimens taken from primary
malignant melanomas (7 cases) and from lymph node metastasis of
malignant melanomas (3 cases), abundant MIA transcripts were amplified.
From all of these specimens, -actin cDNA was coamplified to
control for equivalence and integrity of RNA preparations. In summary,
we detected high levels of MIA mRNA in all malignant melanoma biopsies
and cell lines, low or moderate MIA mRNA levels in most benign
melanocytic nevi, and very low or no MIA mRNA in non-neoplastic skin
biopsies, melanocytes, fibroblasts, and keratinocytes. In addition, we
did not detect any MIA mRNA in a panel of normal mouse tissues
including skin, spleen, brain, thymus, kidney, intestine, lung, and
skeletal muscle (Fig. 4B).
Figure 4:
A, amplification of MIA cDNA in human
biopsies of normal skin, benign melanocytic nevi, and malignant
melanomas. Control PCRs for -actin mRNA are shown in the lower
panel. B, analysis of MIA expression in normal murine
tissues.
Figure 5: Genomic sequence located 5` adjacent to the MIA protein translation start (ATG). The mRNA initiation site is marked by an arrow. Underlined are consensus binding sites for SP-1(-108), NF-kB(-207), CTF/NF-1(-625), a purine-rich sequence (-753 to -731), and an Alu repeat (-1386 to -1215).
To determine whether the MIA promoter is activated specifically in melanoma cells, we cloned the 1386-bp fragment shown in Fig. 5in front of a promoterless CAT plasmid and tested its activity in several human melanoma cell lines in comparison to non-melanocytic cancer cells. We found that the MIA promoter confers high levels of gene expression specifically in human or murine melanoma cell lines but not in HeLa, HepG2, PA-1, and COS cells (Fig. 6, A and B). To map in more detail cis-regulatory elements mediating MIA mRNA expression in melanoma cells, we transfected a series of 5`-deleted CAT reporters both into B16 and COS cells. B16 cells were chosen for this experiment because they were transfected much more efficiently, and therefore small changes in promoter activity could be monitored reliably. Fig. 6B gives a summary of the promoter constructs and CAT activities obtained from transiently transfected B16 and COS cell cultures. Maximal CAT activity was observed when a promoter fragment ranging from -493 to -1 with respect to the ATG protein start codon was used. This promoter fragment conferred 14-fold activation to the basal CAT plasmid in B16 cells in comparison to the Rous sarcoma virus-LTR that conferred 17-fold activation. Significant changes in CAT activities were observed when a series of fragments extending further 5`-upstream was analyzed, indicating that silencer and enhancer elements are located between residues -1200 and -761 and -761 and -493. Further deletion of the promoter to -212 decreased significantly CAT activities, and a promoter fragment starting at -170 was entirely inactive as compared with the promoterless pBLCAT3 plasmid. In summary, these CAT assays led to the conclusion that residues located between -493 and -1 in the MIA promoter are necessary and sufficient to mediate high levels of cell type-specific gene expression.
Figure 6: A, MIA promoter activity in human melanoma, epithelial, and undifferentiated cells. The 1.38-kb MIA promoter fragment was cloned into the promoterless pBLCAT3 plasmid and transfected into human melanoma cells (1144, MelEi, Mel Juso, SK Mel-28) and into HeLa, HepG2, and PA-1 cells. Shown are CAT activities relative to mock-transfected controls. B, deletion analysis of the MIA promoter. Indicated at the left are MIA promoter fragments ranging from residues -1361 to -1. CAT activities resulting from transient transfections into B16 melanoma cells (open bars) or COS cells (shadowed bars) are indicated at the right. Basal activity resulting from the promoterless CAT3 plasmid was set arbitrarily at 1. Values indicate the average of at least three independent transfections.
Figure 7: A, gel mobility shift analysis of the MIA NF-kB site. The phospholabeled binding site was mixed with albumin (lanes 1 and 2) or B16 nuclear extracts (lanes 2-4 and 6-9). Competition experiments were performed by adding 25- or 50-fold excess of the same binding site (MIA-NFkB-Oligo) or 25-fold excess of an unrelated binding site of the MIA promoter (170-Oligo), a mutated MIA-NF-kB binding site (mut NFkB-oligo), or a consensus NF-kB binding site (consensus NFkB-Oligo). See ``Experimental Procedures'' for sequences of binding sites. B, functional analysis of the MIA-NF-kB binding site. pBLCAT3 reporter plasmids under control of the entire 1361-bp MIA promoter (Cat3-MIA) or under control of the promoter mutated at four nucleic acid residues in the NF-kB site (Cat3-MIA-NFkBmut) were transfected into B16 melanoma cells (left) or COS cells (right). Activities are shown from cells treated with PMA (+PMA) or with solvent alone.
NF-kB activity results from a gene family that is expressed in a large number of different cell types and tissues, and consequently the MIA-NF-kB site was also shifted when COS cell extracts were used (data not shown). To address whether binding of the NF-kB site is necessary for cell type-specific function of the MIA promoter, we introduced the same four bases that abolished binding of NF-kB in gel shift experiments by site-directed mutagenesis in the CAT reporter plasmid under the control of the full 1361-bp MIA promoter. This promoter construct mutated at the NF-kB site was transiently transfected into B16 melanoma cells in parallel with the wild-type promoter construct. As shown in Fig. 7B, we observed approximately 2-fold decreased activity in comparison to the wild-type promoter, whereas the mutation did not affect significantly the stimulating effect of PMA on CAT expression. These results indicate that the NF-kB contributes to the MIA promoter activity in melanoma cells but is dispensable for stimulation in response to PMA.
To test whether the induction by PMA represents a primary response or a late event, we determined the time course of mRNA induction and promoter activation. RT-PCR analyses of HeLa and COS cells revealed that the mRNA was first detected 8 h after the onset of PMA treatment. These results were in good agreement with CAT activities obtained from HeLa and COS cells transfected transiently with the MIA promoter-CAT plasmid and treated with an inhibitor of RNA synthesis at various points after PMA induction. Stimulation of CAT activity was not observed when actinomycin D was added to the cell cultures earlier than 9 h after PMA, indicating that it does not represent a primary response (data not shown).
Here, we report the molecular cloning of the human genomic MIA locus, describe its organization, and provide an initial
characterization of cis-regulatory elements within the 5`-genomic
region mediating high levels of gene expression in melanoma cells. The
complete exon-intron structure was determined by sequencing two
adjacent genomic XbaI fragments that cover four small exons
interrupted by three intervening introns. The coding nucleic acid
residues matched perfectly to the cDNA sequence obtained recently from
a malignant melanoma cDNA library (EMBL Library accession no. X75450).
The 5`-mRNA start was mapped by a primer extension experiment and was
further cloned by RACE-PCR using poly(A) RNA from Mel
Im melanoma cells. These experiments revealed that the mRNA is
initiated 70 bases upstream from the protein coding region downstream
of a pyrimidine-rich sequence motif followed by the nucleic acid
residues AC. As frequently observed with TATAA-less genes, the
initiator sequence is flanked 5` by a consensus SP-1 binding site. The
polyadenylation signal AAATACAA is located 43 bases 3` downstream from
the protein stop codon. Assuming a tail of approximately 200-250
adenines, the sizes of the predicted transcript and the mRNA
(approximately 750 bases) observed on Northern blots (Blesch et
al., 1994) are in good agreement.
Data summarized in Table 1and Table 2and Fig. 3and Fig. 4indicate that MIA mRNA expression parallels closely the
malignancy of pigmented skin tumors and is not expressed in normal
tissues of adult mice. By means of RT-PCR results, we detected no or
very little MIA mRNA in non-neoplastic skin biopsies, moderate levels
in the majority of non-malignant melanocytic nevi, and very high levels
in every biopsy from malignant melanomas or metastases from melanomas.
Interestingly, the two skin biopsies that expressed very low levels of
MIA mRNA were taken from sun-exposed facial skin, and therefore MIA
expression might result from subtle activation of melanocytes not
detectable on microscopic examination. In the small number of biopsies
examined in this study, we were not able to correlate levels of MIA
mRNA in benign melanocytic nevi with a certain histological type of
nevi. Therefore, it will be necessary to explore in a larger study
whether MIA expression provides a prognostic parameter to define nevi
at risk for malignant progression. Analyses of other S100 positive
tumors including astrocytomas, oligodendrogliomas, and glioblastomas
indicate that MIA expression is highly associated with melanocytic
tumors and can be detected only occasionally in other neuroectodermally
derived tumors (Blesch et al., 1994). ()
The
melanoma-associated expression pattern of MIA was further substantiated
by RT-PCR amplifications and Northern blot analyses of cell cultures in vitro. Together with data published previously (Blesch et al., 1994), we have now tested 10 different malignant
melanoma cell lines, every one of which expressed very high levels of
MIA mRNA. In contrast, all cultures of non-neoplastic skin cells
including fibroblasts, keratinocytes (HaCaT cells), or melanocytes did
not express MIA mRNA. The close correlation between MIA expression and
melanocytic tumors or tumor cell lines raises questions about the
function of MIA in regulating growth and invasion of malignant
melanomas in vivo. We have observed recently that treatment of
malignant melanoma cells with purified MIA protein in vitro results in growth inhibition paralleled by a significant change in
cell morphology (Apfel et al., 1993; Blesch et al.,
1994). Melanoma cells round up within 2 h after the addition of MIA
protein to the tissue culture supernatant. It is therefore possible
that secretion of MIA in vivo leads to decreased adhesiveness
of melanocytic cells and thereby promotes melanoma progression and
invasion. Ambivalent functions in regulation of tumor cell growth,
invasion, and metastasis have been described for a number of different
signal molecules including tumor necrosis factor- and tumor growth
factor-
(Orosz et al., 1995; Rodeck, 1993). Clearly, more
functional studies are needed to assess the effect of MIA protein on
melanoma cell growth and progression in vivo and further to
define its physiological role in non-neoplastic cells.
Although our molecular analyses of the MIA promoter are still preliminary, our experiments point to a region of less than 500 base pairs, which is sufficient to mediate high levels of gene expression in malignant melanoma cells and which is much less active in benign pigmented skin tumors or normal skin. It will be important to define sites of specific protein interaction within this promoter region to elicit transcriptional changes associated with progression from benign melanocytes to malignant melanoma cells. A number of genes specifically expressed in melanocytes or melanoma cells have been described recently including melanotransferrin (Duchange et al., 1992), tyrosinase (Ganss et al., 1994), and MART-1 (Kawakami et al., 1994), and a promoter fragment of the tissue plasminogen activator gene mediating expression in melanoma cells has been identified (Fujiwara et al., 1994). A careful comparison of the melanocyte-specific promoter regions in these genes did not reveal any obvious cis-regulatory element in common with the MIA promoter.
Another very interesting finding is the activity of an NF-kB-dependent cis-regulatory element present in the MIA promoter. NF-kB is a key mediator of a broad spectrum of signal molecules involved in inflammatory processes, and therefore NF-kB-mediated activation of MIA gene expression is likely to occur in response to the host immune defense. It is well known that melanoma cells frequently elicit a strong inflammatory host response at their site of invasion (Rodeck, 1993; Böcker et al., 1988), and partial regression of melanomas in areas of inflammatory infiltration is a quite common finding. These data point to a molecular link between classical host immune mechanisms in tumor rejection and specific regulation of growth regulatory genes in melanoma cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X84707[GenBank].