©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tissue-specific Enhancer-Promoter Interactions Regulate High Level Constitutive Expression of Matrix Metalloproteinase 2 by Glomerular Mesangial Cells (*)

(Received for publication, December 16, 1994; and in revised form, June 5, 1995)

Sigrid Harendza (§) Allan S. Pollock (§) Peter R. Mertens David H. Lovett(§)(¶)

From theDepartment of Medicine, Department of Veterans Affairs Medical Center, San Francisco, California 94121 and the University of California, San Francisco, California 94123

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Matrix metalloproteinase 2 (MMP-2, (^1)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-beta. 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-beta, 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.


EXPERIMENTAL PROCEDURES

Isolation and Characterization of Rat MMP-2 Genomic Clones

A rat DASHII genomic library (Stratagene) was screened with a 250-bp cDNA probe that represented the 5` proximal end of the cloned rat MMP-2(13) . Library screening and analysis of phage clones were performed using standard methodology. Two overlapping phage clones, 12.1.3 and 18.2.3, were obtained and restriction-mapped using partial digestion with BglII, KpnI, and PstI, followed by Southern blot analysis with oligonucleotide probes for the 5` and 3` ends of the genomic insert. A 6-kb KpnI-NotI fragment containing 500 bp of the second intron and 1686 bp of the 5`-flanking region was subcloned into pBluescript KS+ (Stratagene) and sequenced using a Sequenase version 2 kit (U. S. Biochemical Corp.) and synthetic oligonucleotide primers synthesized on an Applied Biosystems DNA synthesizer.

Luciferase Reporter Constructs

The subcloned 6-kb KpnI-NotI fragment was used as a template to prepare a series of 5`-flanking region reporter constructs in the promoterless luciferase expression vector, pGL2-Basic (Promega). The 5` PCR primers included a KpnI site; the 3` PCR primers included a BglII site to permit directional subcloning into the polylinker region of pGL2-Basic. Initial constructs included sequences extending 1686, 1007, 537, 383, 321, 267, and 239 bp 5` relative to the translational start site. Further fine mapping of enhancer activity was performed by the preparation of sequential deletion constructs extending from bp 1686 to bp 1181 5`. To assess enhancer activity in the context of a fixed heterologous promoter, fragment -1342 to -1262 bp was subcloned in a normal and reversed orientation into the expression vector pGL2-Promoter (Promega), which contains a heterologous SV40 core promoter 5` to the luciferase coding region. To assess distance relationships, the same fragment was subcloned into a BamHI site of the pGL2-Promoter vector, which was located 2000 bp from the SV40 promoter.

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.

Cell Culture and Transient Transfections

The isolation, characterization, and maintenance of rat glomerular mesangial and epithelial cells have been described in detail(11, 12, 13, 14, 15) . The human monocytic leukemia cell line U937 was obtained from the American Type Collection. All cells were maintained in RPMI 1640 medium supplemented with 1% non-essential amino acids, 2 mM glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin, and 10% fetal bovine serum. Mesangial cells and epithelial cells were transfected with Lipofectin (Life Technologies, Inc.) according to Felgner et al.(16) . Lipofectin (10 µl) was diluted in 90 µl of RPMI 1640 medium supplemented with 10% NU serum and mixed with 1 µg of purified plasmid DNA. As control for transfection efficiency, the vector pRSV-betaGal was used at 2.5 µg/100 µl of transfection mixture. The DNA-liposome mixtures were incubated for 10 min at room temperature, after which 800 µl of NU serum-containing medium was added. Cells at 60-70% confluence on six-well culture plates were washed twice with phosphate-buffered saline, and 1 ml of the liposome mixture was added/well. After incubation for 18 h at 37 °C, the liposome mixture was removed and replaced with normal culture medium. Cells were harvested after another 24-h incubation period.

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-betaGal 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 10M. Cells were harvested for assay 48 h following transfection.

Luciferase assay was performed according to Brasier et al.(17) , using a Monolight 2010 luminometer. beta-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%).

Preparation of Nuclear Extracts

Cells of each studied type were grown to 90% confluence in 150-mm tissue culture dishes and nuclear extracts prepared according to Dignam(19) . A 1 M potassium chloride-containing buffer was used for extraction, and the resultant proteins were dialyzed overnight in 20 mM Hepes, pH 7.9, 20% glycerol, 100 mM potassium chloride, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride. The protein concentrations were determined by the BCA protein assay (Pierce) using bovine serum albumin as a standard.

Electrophoretic Mobility Shift Assays

The experiments were carried out according to Carthew et al.(20) . Synthetic or PCR-derived oligonucleotides were digested with BglII and the resultant overhanging 5` ends filled in with [alpha-P]dCTP (3000 Ci/mmol) by means of the Klenow fragment of DNA polymerase I. Binding reactions included 1 10^4 cpm of labeled DNA in a 12.5-µl reaction mixture containing 2 µg of poly(dI-dC), 300 µg/ml acetylated bovine serum albumin, 20% glycerol, and 5 µg of nuclear extract in dialysis buffer. After 15 min at 30 °C, the samples were loaded onto a pre-electrophoresed 4% polyacrylamide (40:1 acrylamide:bisacrylamide), 15% glycerol gel which was polymerized on Gelbond (FMC) and electrophoresed in a buffer containing 1 Tris borate-EDTA. Electrophoresis was conducted at 35 mA for 2.5 h. The gels were air-dried and autoradiographed. For competition experiments, unlabeled DNA fragments were added in 5-500 M excess to the reaction mixture.

DNA Fragments for Gel Shift Experiments

DNA fragments were prepared either by direct synthesis or by PCR(21) . After synthesis, oligonucleotides were purified by electrophoresis on a 10% polyacrylamide, 8 M urea gels, eluted, and precipitated with ethanol. Equimolar quantities annealed to form the final product. Restriction enzyme recognition sites were included at each end to allow labeling by digestion and fill-in with labeled nucleotides using the Klenow fragment of DNA polymerase. Labeled fragments were purified on non-denaturing 10% polyacrylamide gels.

Northern Blot Analysis

Poly(A) RNA (5 µg/lane) from confluent rat MC, confluent and subconfluent rat GEC, and non-stimulated and PMA (10M) differentiated U937 cells were electrophoresed an a 1.0% denaturing agarose gel and transferred to nylon membranes. A 2.7-kb rat MMP-2 cDNA insert (13) was labeled by random hexamer priming with [alpha-P]dCTP. As a normalization control, a full-length human beta-actin cDNA probe was used. Hybridization was performed under standard conditions, followed by a high stringency wash in 1% SDS, 0.1 SSC at 65 °C for 60 min.

DNA Footprinting Analysis

The oligonucleotide probe for footprinting was prepared by digestion of construct pT4-Luc 1342P with Asp 718, which created a 5` overhang at the KpnI site of the non-coding strand. The overhang was filled in with an excess of [alpha-P]dGTP (6000 Ci/mmol) using Klenow fragment, thereby resulting in an end-labeled coding strand. Labeling was followed by BglII digestion and the mixture was loaded on a 8% polyacrylamide gel (40:1 acrylamide:bisacrylamide) for separation of the 80-bp insert from the vector. After electrophoresis the gel was exposed to Kodak XAR-2 film for 1 min, and the labeled 80-bp fragment was cut out, extracted from the gel piece with 0.5 M ammonium acetate for 3 h, and ethanol-precipitated overnight.

The DNA-protein mixture was prepared as described for the gel retardation assay, but 10^5 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(2)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.

Transcription Start Site Mapping

Fifteen micrograms of poly(A) RNA was prepared from mesangial cells and hybridized with 2 pmol of a 35-mer oligonucleotide, which was end-labeled using polynuclotide kinase and [-P]ATP (6000 Ci/mmol). The primer was designed to hybridize to the predicted MMP-2 transcript approx100 nucleotides 3` to the analogous human MMP-2 mRNA transcription start site. The primer was extended using avian myeloblastosis virus reverse transcriptase by standard techniques. The products were resolved on an 8% acrylamide, 7 M urea sequencing gel. A sequencing ladder was prepared using the same labeled oligonucleotide to sequence a plasmid containing the genomic clone with the predicted start site. It was resolved on the same gel used for the primer extension products.

Sequence Analysis

The assembled sequence was compared with the Transcription Factor Database version 7.3 (23) using the Quest program of IntelliGenetics Suite (IntelliGenetics, Mountain View, CA), or using the FASTA program(41) .


RESULTS

Characterization of the Proximal 5` Regulatory Region of the Rat MMP-2 Gene

For the initial investigation of the 5` regulatory region of the rat MMP-2 gene, a 17-kb clone (clone 12.1.3) was isolated from a rat phage genomic library using a cDNA probe that hybridized within exon 1. Restriction mapping of clone 12.1.3 yielded a 6-kb KpnI-NotI fragment (NotI from the DASHII vector), which extended from the second intron at the 3` end, through exons 1 and 2, and included 1686 bp 5` of the initiator methionine. The KpnI-NotI fragment was subcloned into pBluescript KS+ and sequenced. The sequence is shown in Fig.1A, beginning 1686 bp upstream of the translation initiation codon. In contrast to other known members of the MMP supergene family, there were no TATA or CAAT boxes, as is the case with the human MMP-2 gene(1, 4, 5, 7) . Further similarity with the human MMP-2 gene is manifested by the presence of two GC boxes (Sp1 binding sites) immediately adjacent to the putative transcriptional start site based on the human sequence. A potential AP-1 element was present within the first exon, whereas three AP-2-like elements, which differed from the consensus sequence (CCCCAGGC) by 2-3 nucleotides, were present within the 5`-flanking sequence. A 49-bp potential Z-DNA sequence was also present(24, 25) , and five -interferon response elements were identified within this region.


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.

Transcriptional Activity of the Rat MMP-2 Regulatory Region in MC

To identify the sequence requirements for MMP-2 gene transcription by rat MC, cells were transiently transfected with a set of 5` deletion constructs, which contained variable lengths of upstream sequence subcloned into a promoterless luciferase expression vector (Fig.2A). Co-transfection with a beta-galactosidase-expressing plasmid was performed to normalize for differences in transfection efficiency. A moderate increase in luciferase activity was observed with constructs increasing in length up to 1007 bp. With construct pT4-Luc 1686, which contains the whole 5` sequence of the original KpnI-NotI restriction fragment, a major increment in transcription activity was obtained. This suggested that an important cis-regulatory element is present within this part of the sequence. Therefore, in a second set of 5` deletion constructs, a stepwise removal of the 5` end of construct pT4-Luc 1686 was carried out and these constructs were also transfected into rat MC (Fig.2B). High luciferase expression was achieved with constructs that lack up to 344 bp of the 5` end (construct pT4-Luc 1342). Further removal of another 80 bp resulted in a loss of about 80% of luciferase activity (construct pT4-Luc 1262).


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 beta-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.



Identification of an 80-bp Region Containing MMP-2 Enhancer Activity

As described, the removal of an 80-bp fragment spanning -1262 bp to -1342 bp of the 5` regulatory region abolished more than 80% of transcription activity from luciferase construct pT4-Luc 1342. When this 80-bp region was cloned into the luciferase vector pGL2-Promoter upstream of a heterologous SV40 promoter, it was found to enhance transcription 4-fold (Fig.3). We demonstrated orientation and position independence by subcloning this 80-bp fragment into the luciferase plasmid with the SV40 core promoter in the opposite orientation adjacent to the promoter (construct pT4-Luc 1342PI) and in the correct orientation at a distance of approximately 2000 bp upstream from the promoter (construct pT4-Luc 1342PD). Compared to the construct pT4-Luc 1342P that contains the 80-bp fragment in the correct orientation next to the SV40 promoter, constructs pT4-Luc 1342PI and pT4-Luc 1342PD show similar levels of luciferase expression. These data indicate that the 80-bp fragment behaves with characteristic enhancer properties. Notably, the 80-bp sequence immediately 3` to this region (construct pT4-Luc 1262P) demonstrated no transcriptional enhancing activity.


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 beta-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.



Nuclear Protein Binding to MMP-2 Enhancer Region

Gel mobility shift analysis was performed with nuclear extracts from MC to identify protein-interaction(s) with the 80-bp fragment pT4-1342P enhancer. Fig.4shows a gel shift assay carried out with this fragment radiolabeled with [alpha-P]dCTP. Fragment pT4-1342P and also the immediate 5` fragment pT4-1262 were used as non-radioactive competitors in 0.1-100-fold molar excess. In the presence of mesangial nuclear extract the mobility of the radiolabeled 80-bp fragment is significantly retarded (lane2) and reveals a major DNA-protein complex compared to the mobility of the unbound DNA fragment alone (lane1). Specificity of this interaction is demonstrated in lanes 3-6, where addition of increasing amounts of cold fragment pT4-1342P to the DNA-nuclear extract mixture successfully competes for the binding protein(s), resulting in the disappearance of the shifted band. The specificity of this DNA-protein interaction is further confirmed by the fact that a different non-radioactive fragment (pT4-1262P) was unable to compete for protein binding (lanes 7-9). Additional minor shifting bands were evident but not investigated further because of the very small fraction of probe shifted.


Figure 4: Gel shift analysis performed with [alpha-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 alpha-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 beta-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.



Mutational Analysis of Enhancer Region

Finer mapping of the 40-bp sequence of pT4-1342B-P, which contained both DNA binding and transcriptional activity, was performed by regional mutation of the sequence. Three mutant 40-bp sequences were prepared from complimentary oligonucleotides, each containing 9 non-complementary mutated bases (underlined) in different regions of the 40-bp sequence (see Fig.8A for exact locations). Pyrimidines were replaced by their non-complementary purines and vice versa. This resulted in the mutants pT4-1342B-Palpha, with the mutation in the 5` part of the 40-bp sequence; pT4-1342B-Pbeta, with the mutation in the middle part; and pT4-1342B-P, which was mutated at the 3` end. A gel shift analysis with the non-mutated alpha-P-labeled 40-bp fragment pT4-1373B-P and the radiolabeled mutants is shown in Fig.8A. Mutation of 9 bases at the 5` end of the 40-bp fragment (pT4-1342B-Palpha) and mutation at the 3` end (pT4-1342B-P) resulted in a normal gel shift of the major DNA-protein complex, when compared with the shift of the non-mutated 40-bp fragment. There was no change in the relative mobilities of the DNA-protein complex with these oligonucleotides. However, the oligonucleotide, pT4-1342B-Pbeta, with the mutated area in the middle of the 40-bp fragment shifted only minimally. When the non-mutated 40-bp fragment was used as radiolabeled probe and the non-mutated and mutated fragments were added as competitors in 50-fold molar excess (lanes 4-8), only the mutants pT4-1342B-Palpha and pT4-1342B-P competed successfully for protein binding, whereas mutant pT4-1342B-Pbeta did not show any competition. Thus, the sequence within 1342B with protein binding properties is contained within the 11-bp palindromic sequence.


Figure 8: Gel shift analysis with alpha-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 alpha-P-labeled mutants pT4-1342B-Palpha (lane2), pT4-1342B-Pbeta (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-Palpha (lane6), pT4-1342B-Pbeta (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-Palpha, pT4-Luc 1342B-Pbeta, and pT4-Luc 1342B-P as enhancers. Transfection efficiency was evaluated by normalization with beta-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-Palpha, which showed a normal gel shift, did not demonstrate any transcriptional activity. The mutated middle sequence (pT4-1342B-Pbeta) also did not act as an enhancer. Mutated fragment pT4-1342B-P, which extends beyond the footprinted region, showed normal transcriptional activity.

Tissue-specific Expression of the MMP-2 Gene

Cell-specific expression of the MMP-2 gene was investigated in cell lines of diverse embryological origins, including glomerular mesangial cells (MC), glomerular epithelial cells (GEC), and monocytic leukemia U937 cells. MC in culture secrete large quantities of MMP-2(10) ; in contrast, GEC derived from adult animals do not secrete this enzyme(14) . Non-differentiated U937 cells also fail to secrete MMP-2, but do so after differentiation is induced with phorbol esters(3) . As shown by Northern blot analysis (Fig.9A), cultured MC have a high abundance of the mRNA transcript for MMP-2. The MMP-2 mRNA was present to a lesser extent in differentiated U937 cell, whereas non-stimulated U937 cells had no detectable MMP-2 transcripts. Neither subconfluent nor confluent glomerular epithelial cells had detectable MMP-2 mRNA.


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 beta-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 beta-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.

Tissue Specificity of the 80-bp Enhancer Element

For the investigation of cell specific activity of the 80-bp MMP-2 enhancer, gel shift analysis was performed with nuclear extracts prepared from all cell lines described above (Fig.10A). The gel shift analysis revealed in each case a single shifted band with the same relative mobility as observed from MC nuclear extracts before. Transfection of the individual cell types with construct pT4-Luc 1342P, which contains the 80-bp enhancer in context with the heterologous SV40 promoter, is demonstrated in Fig.10B. Although high transcriptional activity is seen in MC, no activation of transcription can be found within non-stimulated or stimulated U937 cells, which both show protein binding to the enhancer region in the gel shift assay. In contrast, transcription was stimulated over 8-fold when this construct was used to transfect GEC, which do not secrete MMP-2, but which show protein binding to the enhancer region in the gel shift analysis.


Figure 10: A, gel shift assay performed on alpha-P-labeled 80-bp fragment pT4-1342P with 5 µg of nuclear proteins from MC (MC), non-stimulated U937 cells (U937), PMA (10M) 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 beta-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.



Identification of Tissue-specific Promoter Activity

The above findings suggested that cellular specificity of transcription does not simply depend on (a) the presence or absence of the 80-bp enhancer binding protein and (b) transcriptional activity within the context of a heterologous promoter. These results suggested that additional levels of transcriptional control rested within the intrinsic MMP-2 promoter. To test this hypothesis, a heterologous CMV immediate-early enhancer was placed 5` to MMP-2 proximal promoter constructs ranging in size from 239 to 383 bp relative to the transcriptional start site (Fig.11). Transfection into MC yielded major increases in luciferase activity with construct pT4-Luc 321-CMV and pT4-Luc 383-CMV, indicating that a minimal promoter was present between -267 and -321. None of these constructs exhibited significant transcriptional activity in GEC or U937 cells, which indicates that the proximal MMP-2 promoter responsible for transcription in MC is not operative in these cells, or that other cis-acting elements in the MMP-2 gene are necessary for transcription.


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 beta-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.




DISCUSSION

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-kappaB 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.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants DK 39776 and DK 31398 and by Grants Ha 2056/1-1 (to S. H.) and Me 1365/1-1 (to P. R. M.) from the Deutsche Forschungsgemeinschaft. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U30822[GenBank].

§
These authors contributed equally to this work.

To whom all correspondence should be addressed: VA Medical Center, 111J, 4150 Clement St., San Francisco CA 94121. Tel.: 415-750-2032; Fax: 415-750-6949.

^1
The abbreviations used are: MMP, matrix metalloproteinase; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; CMV, cytomegalovirus; PMA, phorbol 12-myristate 13-acetate.


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