(Received for publication, December 16, 1994; and in revised form, June 5, 1995)
From the
The 72-kDa gelatinase A (MMP-2) is a central mediator of the response of the intrinsic glomerular mesangial cell to inflammatory stimuli and is regulated in a unique, cell-specific manner. We isolated a 6-kilobase pair genomic fragment of the rat MMP-2 gene and sequenced and characterized 1686-base pair of the 5`-flanking region. Using a series of 5` deletion constructs of the proximal 5`-flanking region, a strong MMP-2 enhancer element was identified. Gel shift and mutational analyses suggest that the enhancer region represents the binding site for a complex transcription factor demonstrating separable DNA-binding and transcriptional activating domains. The presence and activity of the enhancer element was evaluated in several cell types with varying capabilities to synthesize MMP-2 including mesangial cells, glomerular epithelial cells, and the monocytic U937 cell. Although binding activity was present in all cell types studied, enhancer activity was demonstrated only in mesangial and glomerular epithelial cells. Additional transcriptional control resided in a tissue-specific promoter, which supported transcription only in mesangial cells. These results indicate that the final control of mesangial cell-specific synthesis of MMP-2 derives from an interaction between the strong enhancer element and the tissue-specific MMP-2 promoter.
Matrix metalloproteinase 2 (MMP-2, ()also known as
the 72-kDa gelatinase A) is a member of the matrix-degrading family of
metalloproteinases, which are characterized by activity at neutral pH,
dependence upon zinc for catalytic activity, secretion in a latent,
proenzyme form, and inhibition by the tissue inhibitors of
metalloproteinases. Other members of this gene family include the
extensively characterized interstitial collagenase, stromelysin,
matrilysin, and the 92-kDa gelatinase B(1) . Although both the
72-kDa (MMP-2) and the 92-kDa (MMP-9) type IV collagenases have similar
substrate specificities(2, 3) , their patterns of
expression are different. Correspondingly, their 5`-flanking regions
that have been reported to date bear few
similarities(4, 5, 6) .
In most cases, the
synthesis of MMP-2 by non-transformed or tumor cell lines is
constitutive in nature, at least in terms of responsiveness to phorbol
esters, inflammatory cytokines, and transforming growth factor-.
The lack of responsiveness to such reagents has been attributed to the
absence of AP1, PEA3, TIE, and other regulatory sequences within the
5`-flanking region of the MMP-2 gene. These elements are commonly found
within the flanking regions of other members of this gene
family(7) . However, tissue-specific and developmentally
related control of MMP-2 clearly exists in vivo and in some in vitro experimental systems. For example, MMP-2 is highly
expressed in the developing murine lung and kidney, with minimal
expression in bone, or the central nervous system (8) . Upon
completion of organ development, MMP-2 expression is minimal. Monocytic
cells also show evidence for differentiation-specific expression of
this enzyme, as progression toward a macrophage phenotype is associated
with inducible MMP-2 synthesis(9, 10) .
MMP-2 is a
key component of the basal turnover of the renal glomerular basement
membrane(11) . Additionally, enhanced synthesis of MMP-2 during
glomerular inflammatory disorders plays a major role in the evolution
of the disturbances of renal glomerular extracellular matrix structure
and function that are characteristic of these
disorders(11, 12) . In recent studies we have
demonstrated that glomerular mesangial cell regulation of MMP-2
synthesis exhibits a number of unique features, including stimulation
by interleukin-1/tumor necrosis factor, transforming growth
factor-, phorbol esters, and cAMP
analogs(12, 13) . Given this distinguishing pattern of
regulation, we have investigated the transcriptional regulation of the
proximal 5`-flanking region of the rat MMP-2 gene within several
distinctive cellular contexts. The current study demonstrates that a
complex pattern of cell type-specific regulation of MMP-2 synthesis
occurs, which involves cell type-specific cooperative interactions
between a strong enhancer element and the proximal promoter of this
gene.
To study intrinsic MMP-2 promoter activities within the context of a heterologous enhancer, sections of the proximal MMP-2 promoter extending from the translational start site to -239 to -383 bp were subcloned into the pGL2-Basic vector, which also contained the 550-bp CMV immediate/early enhancer. As a positive control the CMV enhancer was also subcloned into the BamHI site of the pGL2-Promoter vector.
U937 cells were
transfected by electroporation in calcium/magnesium-free
phosphate-buffered saline using a Bio-Rad electroporator set at 500
microfarads and 290 V, using 10 µg of construct DNA and 15 µg
of pRSV-Gal DNA. After electroporation, the cuvettes were
incubated on ice for 15 min and 250 µl of the cell mixture were
added to 3 ml of growth medium. For experiments with differentiated
U937 cells, phorbol myristoyl acetate (PMA) was added 5 h after
electroporation to a final concentration of 1
10
M. Cells were harvested for assay 48 h following
transfection.
Luciferase assay was performed according to Brasier et al.(17) , using a Monolight 2010 luminometer.
-Galactosidase activity was determined as reported and used to
normalize for transfectional efficiency(18) . All transfections
were performed in triplicate to quadruplicate for each point, and
transfections were repeated at least three times. Transfection results
were averaged and are expressed as the means (S.D. less than 15%).
The DNA-protein mixture was prepared as described for the gel
retardation assay, but 10 cpm of the probe were used per
reaction. After incubation for 15 min at 30 °C, 2 µl of a 1:10
dimethyl sulfate solution were added for 30 s and the reaction was
terminated by addition of 2 µl of 1 M
dithiothreitol(22) . The samples were then separated by
electrophoresis on a 4% polyacrylamide gel (40:1
acrylamide:bisacrylamide). The gel was exposed to a Kodak XAR-2 film
for 10 min, and the shifted and non-shifted bands were identified and
excised. Methylated DNA was extracted with 0.5 M ammonium
acetate for 3 h and precipitated overnight with ethanol at -80
°C after a phenol/chloroform extraction. The dried pellets were
resuspended in 45 µl of H
O and incubated with 5 µl
of 10 M piperidine for 30 min at 90 °C to cleave the DNA.
The reaction was precipitated with ethanol and 125 µg/ml tRNA
overnight at -80 °C and extensively dried under vacuum.
Thereafter the cleaved DNA was resuspended in 10 µl of 80%
formamide and 0.2% bromphenol blue. Equivalent amounts of radioactivity
of the shifted and non-shifted DNA were loaded onto a 8%
polyacrylamide, 7 M urea, TBE sequencing gel. The gel was
electrophoresed at 65 watts constant power until the dye reached the
lower third of the gel, then transferred to Whatman No. 3MM paper,
dried under vacuum and heat, and exposed to a Kodak XAR-2 film with an
intensifying screen overnight at - 80 °C.
Figure 1:
A,
nucleotide sequence of the rat MMP-2 gene extending 1686 nucleotides
upstream of the ATG translation initiation codon. Arrows represent the major and minor start site of transcription as
determined within the human sequence (14) , which shows a 100%
homology to the rat sequence within this region. Boxes indicate potential binding sites for relevant transcription
factors, including AP-1, AP-2, Sp-1, -IRE sites, and a Z-DNA
sequence. B, sequence homology between the rat and the human
proximal promoter regions. Analogous to the limited published human
sequence(14) , there were no TATA or CAAT boxes in the rat
gene. Arrows represent the major and minor transcriptional
start site of the rat MMP-2 gene, which are identical to those of the
human gene. Potential transcription factor binding sites are indicated,
and sites shared by both species are shown in boldface. R, rat; H, human. Nucleotides are numbered beginning at the ATG codon.
A comparison with the limited human MMP-2 5`-flanking sequence (4) revealed a high degree of homology with approximately 65% nucleotide identity (Fig.1B). Within the sequence encompassing the major and minor transcriptional start site of the human sequence, there is 100% homology to the rat MMP-2 sequence. Primer extension analysis of rat mesangial cell mRNA mapped two transcription start sites at -264 and -274 relative to the translation start site. These located in an identical region to those found in the human gene, except that the 5`-untranslated region of the human mRNA is 27 nucleotides longer. An AP-2-like element is found in the 5`-flanking sequence of the rat promoter region, whereas an AP-2-like site is present within the first exon of the human MMP-2 gene. No AP-1 sequences are present within the limited human sequence, whereas the rat sequence reveals a potential binding region within the first exon.
Figure 2:
Analysis of transient transfection
experiments obtained with two sets of 5` deletion constructs in MC.
Nucleotide positions with respect to the start site of translation are
indicated above the line. Data are the ratios of luciferase versus -galactosidase activities, arbitrarily set equal to 100% for
pT4-Luc 1686. Results are means of two to six independent transfection
experiments (done with triplicate to quadruplicate plates) performed
with two different preparations of each plasmid. A, 5`
deletion constructs of the whole 1686-bp sequence. B, stepwise
removal of the 5` end of construct pT4-Luc 1686 from -1686 to
-1187 bp.
Figure 3:
Transcriptional activity of the 80-bp
fragment pT4-1342P in MC. This 80-bp fragment of the 5` region of the
MMP-2 gene was subcloned into pGL2-Promoter, a SV40 promoter-containing
luciferase plasmid, resulting in construct pT4-Luc 1342P. Transient
transfections were carried out as described under ``Experimental
Procedures.'' Numbers are given as ratios of luciferase versus -galactosidase activities. The activity of vector
pGL2-Promoter was assigned a value of 100%. Blacklines, pGL2-Promoter sequences; blackboxes with arrow, location and orientation of 80-bp fragment
pT4-1342P within the vector; whitebox, 80-bp control
fragment pT4-1262P.
Figure 4:
Gel shift analysis performed with
[-
P]dCTP-labeled 80-bp fragment (pT4-1342P)
and nuclear extracts from MC. The mobility of the radiolabeled fragment
alone (lane1) and after incubation with nuclear
extract (lane2) are shown. The arrow indicates the mobility of a major DNA-protein complex. Competition
was performed with a 0.1-100-fold molar excess of non-radioactive
fragment pT4-1342P (lanes3-6) or a
0.1-10-fold molar excess of a different non-radioactive fragment
pT4-1262P (lanes 7-9).
A second gel shift analysis was performed for the further mapping of the 80-bp enhancer element (Fig.5). In this experiment, overlapping 40-bp competitor subfragments of the 80-bp fragment pT4-1342P were used in 50-fold molar excess as outlined in the legend of Fig.5. We observed a distinct pattern of competition for nuclear protein using these unlabeled 40-bp competitor fragments. Lane 1 shows the gel shift of the radiolabeled fragment pT4-1342P and lane 2 the specific competition with the cold 80-bp fragment. Although fragment pT4-1342A-P, which spans the 5` 40 bp of pT4-1342P, does not compete for the major binding protein(s) (lane 4), fragment pT4-1342B-P, which corresponds to the middle 40 bp of pT4-1342P, competes successfully for protein(s) in the major shifted band (lane 6). Fragment pT4-1342C-P, the 3` 40-bp region of pT4-1342P, reveals no competition for the major protein(s) (lane 8). Lanes 3, 5, and 7 serve as controls without competitor DNA. These data localize the major gel binding region of the 80-bp fragment pT4-1342P is contained within the 40-bp subsequence pT4-1342B-P. Transfection studies in MC demonstrated that the subsequence of 1342B-P (bp -1322 to -1282), when subcloned into the luciferase expression vector with the heterologous promoter, manifested all the enhancer activity contained within the original 80-bp segment (Fig.6).
Figure 5:
Gel shift experiment performed on
-
P-labeled 80-bp fragment pT4-1342P incubated with 5
µg of crude nuclear proteins from MC (lanes 1-8).
Competition was performed with a 50-fold molar excess of the
non-radioactive 80-bp fragment pT4-1342P (lane2) or
non-radioactive 40-bp fragments pT4-1342A-P, pT4-1342B-P and
pT4-1342C-P (lanes4, 6, and 8,
respectively). Lanes3, 5, and 7 serve as controls with no competing fragments. The shifted
DNA-protein complex is indicated by the openarrow.
Sequences of competition fragments are shown underneath the
picture.
Figure 6:
Transient expression of pGL2-Promoter
constructs pT4-Luc 1262P and pT4-Luc 1342P (both containing 80-bp
inserts) and pT4-Luc 1342A-P and pT4-Luc 1342B-P (both containing 40-bp
subsequences of the 80-bp fragment) in MC. The data are ratios of
luciferase versus -galactosidase activity, arbitrarily
set equal to 100% for the pGL2-Promoter. Blacklines,
pGL2-Promoter vector sequences; whitebox, 80-bp
fragment with no competition in gel shift assay; hatchedboxes, 80-bp and 40-bp fragments with competition
activity in gel shift assay.
A dimethyl sulfate protection footprinting analysis performed on the coding strand of the 80-bp enhancer element localized the site of DNA-protein interaction to the region denoted with the circled G nucleotides (Fig.7). The footprinted region is extends over 26 bp and contains a 11-bp palindromic segment (marked by a line). The sequence involved in specific DNA-protein interaction does not correspond on a search of the Transcription Factor Database version 7.3 to recognition elements of any known transcription factors.
Figure 7: Dimethyl sulfate footprinting analysis of the 80-bp fragment, pT4-1342P, in the absence (lane A) and presence of 5 µg of crude nuclear proteins from MC (lane B). Footprinting was performed on the end-labeled coding strand in combination with gel shift analysis as described under ``Experimental Procedures.'' Protected regions are indicated by circles. A short palindromic sequence within the footprinted region is marked by a line.
Figure 8:
Gel shift analysis with
-
P-labeled mutated oligonucleotides obtained from
fragment pT4-1342P. A, lane1 shows the
40-bp fragment pT4-1342B-P incubated with 5 µg of nuclear extract
from MC, which resulted in one major shifted band (marked by an arrow). Gel shift analysis was also performed with 5 µg of
nuclear extract from MC and
-
P-labeled mutants
pT4-1342B-P
(lane2), pT4-1342B-P
(lane3) and pT4-1342B-P
(lane4). For
competition experiments, 50-fold molar excess of the non-radioactive
40-bp fragment pT4-1342B-P (lane5) and the 40-bp
fragments with mutated bp pT4-1342B-P
(lane6),
pT4-1342B-P
(lane7) and
pT4-1342B-P
(lane8) were used. Nucleotide
sequences of mutants of pT4-1342B-P are shown below the picture. Each
mutant contains 9 mutated bases (underlined) in different
regions of the 40-bp sequence. Pyrimidines are substituted by their
non-complementary purines and vice versa. B,
transient transfection of MC with SV40 promoter constructs pT4-Luc
1342B-P, pT4-Luc 1342B-P
, pT4-Luc 1342B-P
, and pT4-Luc
1342B-P
as enhancers. Transfection efficiency was evaluated by
normalization with
-galactosidase activity. Data are expressed as
ratios of the luciferase constructs versus the parental
pGL2-Promoter vector.
Transfection experiments in MC were performed to evaluate the
enhancer properties of these mutated 40-bp fragments after ligation
into the luciferase expression vector with the heterologous SV40
promoter (Fig.8B). Mutant
pT4-1342B-P, which showed a normal gel shift, did
not demonstrate any transcriptional activity. The mutated middle
sequence (pT4-1342B-P
) also did not act as an enhancer.
Mutated fragment pT4-1342B-P
, which extends beyond the
footprinted region, showed normal transcriptional activity.
Figure 9:
A, Northern blot analysis of
poly(A)-selected RNA (5 µg/lane) from MC,
subconfluent (s) and confluent (c) GEC,
non-differentiated U937 and differentiated (*) U937 cells.
Hybridization at high stringency was performed with a labeled 2.7-kb
rat MMP-2 cDNA probe and a
-actin cDNA probe as control. B, transfection of all cell lines with luciferase construct
pT4-Luc 1686 in the promoterless luciferase expression vector
pGL2-Basic. Luciferase numbers are normalized by
-galactosidase
expression, and data are given as ratios of luciferase-construct versus pGL2-Promoter vector. Lanes are labeled corresponding
to A.
The activities of construct pT4-Luc 1686, which contains the entire 5`-flanking region present in the original KpnI-NotI restriction fragment, were compared in MC, GEC, and non-differentiated and differentiated U937 cells. Transfection of GEC and U937 cells (both basal and PMA differentiated) with pT4-Luc 1686 yielded minimal luciferase activity in these cells as compared to MC. Thus, within the context of the entire 1686-bp 5`-flanking region, cell-specific activity for transcription is demonstrable.
Figure 10:
A, gel shift assay performed on
-
P-labeled 80-bp fragment pT4-1342P with 5 µg of
nuclear proteins from MC (MC), non-stimulated U937 cells (U937), PMA (10
M) stimulated U937
cells (U937*), subconfluent rat GEC (GEC(s)), and
confluent rat GEC (GEC(c)). The major shifted DNA-protein
complex is indicated by an arrow. B,
transient luciferase expression obtained with construct pT4-Luc 1342P
in all cell lines. Cotransfection with pRSV
-galactosidase was
used to normalize the luciferase data for transfection efficiency, and
data are shown as ratios of luciferase-construct versus the
parental vector pGL2-Promoter. Lanes are marked corresponding to A.
Figure 11:
Transient transfections of all cell lines
with fragments pT4-239-383 subcloned into vector pGL2-Basic,
which was substituted with the minimal heterologous CMV enhancer. The
numerical name of the plasmid indicates the number of nucleotides 5` to
the translation start site that were included in the construct. Data
are normalized with -galactosidase activities and expressed as
ratios of luciferase-construct versus construct pGL2-Promoter
containing the CMV enhancer. 1, pT4-Luc 239-CMV; 2,
pT4-Luc 267-CMV; 3, pT4-Luc 321-CMV; 4, pT4-Luc
383-CMV.
In this study the transcriptional regulatory activities present within the proximal 1686 bp of the rat MMP-2 gene were examined within several cellular contexts. This region as a whole demonstrated appropriate high transcriptional activity in MC, which synthesize MMP-2, and no transcriptional activity in GEC, which do not secrete MMP-2. In U937 cells this region did not reveal transcriptional activity, although these cells produce MMP-2 when stimulated with PMA. Two important regions of this segment are informative: first, a unique and complex enhancer region, which supports the majority of transcriptional activity in mesangial cells; and second, a tissue-specific promoter operative only in mesangial cells.
Tissue- or cell-specific regulation of gene transcription, especially genes with highly restricted expression, often involves cooperative interactions between several regulatory elements(26, 27, 28, 29, 30) . Although the simplest system involves the expression of a key regulatory protein by a single cell type, this does not appear to be the case for the regulation of MMP-2, as all studied cell types contained nuclear proteins capable of binding to the protein binding region of the unique MMP-2 enhancer sequence as shown in Fig.10A. Alternatively, the binding to this specific DNA sequence could represent the function of several distinct nuclear proteins, heteropolymers of nucleoproteins, or proteins of differing phosphorylation states, which may not all result in similar transcriptional activation of MMP-2. Cooperative interactions between enhancer elements and their cognate promoters have also been described(31, 32) , and the results obtained in the current study may be consistent with these different models of cell-specific gene regulation.
Within glomerular mesangial cells, which display a unique pattern of MMP-2 regulation(12, 13) , a strong enhancer element was identified between bp -1322 and -1282 relative to the translational start site. DNA-footprinting analysis indicated a region of protein-DNA interaction extending over 26 bp including a 11-bp palindrome comprising the 3` part of this region. Mutational analysis revealed several important features. The region required for DNA-protein interaction in the gel shift assay was entirely contained within the 3` segment of this region and was necessary and sufficient for DNA binding, but not for transcriptional enhancement. The immediately adjacent 5` segment was necessary for transcriptional enhancer activity in combination with the palindromic region, but insufficient by itself for mobility shift or transcriptional enhancement. The length of the protected footprint and the separation of binding and transactivation regions suggest a transcription factor, either as a single protein or a multimeric protein complex, with several DNA binding domains(33) .
This 40-bp region acted to
increase transcription from the heterologous SV40 promoter in MC, which
express MMP-2, and in GEC, which do not. However, this enhancer was
inactive in PMA-induced U937 cells, which do express MMP-2. This occurs
despite the presence of the mobility shifting activity, which migrates
identically in all three cells, albeit in differing amounts. The
ubiquity of mobility shifting activity, but not of enhancer activity,
may be explained in several ways. Cell-specific transcriptional
enhancement might demonstrate a dependence on other DNA sequences
including promoter dependence. Alternatively, although the mobility
shift in each of these cells did not distinguish shifting proteins of
different composition, cell-specific differences in the subunit
composition of a multimeric binding protein as is seen in the NF-B
family(34, 35) , or in its phosphorylation state as is
seen in interferon-regulated genes and lactation-associated
genes(36, 37) , may account for the difference in
activity.
The proximal promoter was localized to a region analogous to that found by Frisch(39) . The promoter also demonstrates tissue specificity. It is active in mesangial cells, had no activity in GEC, which do not express MMP-2, but also failed to manifest transcriptional activity in PMA-induced U937, cells which do express MMP-2. This indicates that other cis-acting regions must be involved in the expression of this protein by monocytic cell types. These could include regions in a downstream intron, or elements further 5`, including a distinct 5` promoter/enhancer(38) . Identification of alternate elements will require examination of both additional gene regions and an examination of the fine-structure of the 5`-most sequence of the MMP-2 mRNA transcripts in different cell types of the same species.
The complexity of cell-specific regulation of MMP-2 synthesis is underscored by the study of Frisch(39) , which represents the only other detailed published analysis of MMP-2 transcriptional regulation. This study was performed within the context of HT1080 cells, a human fibrosarcoma line, which constitutively synthesizes large amounts of MMP-2(2, 39, 40) . The study identified a major enhancer sequence localized at approximately -1650 bp relative to the transcriptional start site. Deletion or mutation of this sequence, which demonstrated binding activity for the transcription factor AP-2, virtually eliminated transcriptional activity within HT1080 cells. In contrast to our own studies detailed above, the region of the human gene from -1635 bp to the proximal promoter was fully dispensable for effective transcription in HT1080 cells. Perhaps the most interesting finding of that study, which was not found in our system, was the delineation of a strong tissue-specific silencer element localized immediately adjacent to the AP-2-like element. This element was shown to repress transcription very effectively in non-MMP-2 producing cells, suggesting that cell type-specific silencing of the human MMP-2 gene may be operative in addition to cell type-specific enhancer/promoter interactions.
The further characterization of the enhancer binding proteins and an analysis of the more distal 5` regulatory regions of the MMP-2 gene may be expected to provide additional insights into the regulation of MMP-2 expression within the context of the mesangial cell, where this critical inflammatory mediator is central to renal disease states.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U30822[GenBank].