THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Val. 264, No. 14, Issue of May 15, pp. 8339-8344,1989 Printed in USA.

Transcriptional Regulation of Human Stromelysin” (Received for publication, September 26, 1988)

Susan Quinones, Juan Sauss, Yoshihide Otani, Edward D. Harris, Jr.8, and Markku Kurkinen From the DeDartrnent of Medicine. Uniuersitv of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 ’

” .

We have determined that human stromelysin mRNA can be induced by interleukin-18 (IL-18) and that the induced mRNA levels can be suppressed by retinoic acid and dexamethasone (Saus, J., Quinones, S. , Otani, Y., Nagase, H., Harris, E. D., Jr., and Kurkinen, M. (1988) J. Biol. Chem. 263, 6742-6745). Here we show, by nuclear run-on and transient gene expression analyses, that IL-18 induction is a promoter function and that dexamethasone suppresses 1L- 18-induced gene activity. For transient gene expression assays, 1.3 kilobase pairs of the stromelysin promoter region (-1303 to -11 relative to the transcription start site) and shorter fragments thereof were cloned into a hu- man growth hormone reporter vector. In transfected human fibroblast cultures all the constructs, with the exception of the one containing the shortest promoter fragment (-53 to -ll), responded to lL-1B induction. Interestingly, the ability of IL-18 to induce human growth hormone expression decreased as the length of the promoter fragment was reduced. Dexamethasone treatment suppressed the induced human growth hor- mone levels by approximately 50% irrespective of the promoter length. These results suggest that the 1.3- kilobase pairs stromelysin promoter fragment contains DNA elements required for IL- 18 induction and dexa- methasone suppression.

Maintenance of the equilibrium between deposition and degradation of extracellular matrix is essential to normal tissue development and repair of wound or inflammatory damage. In the pathological states of rheumatoid arthritis and tumor invasion, degradation is disproportionately increased and the equilibrium destroyed (1-3). Matrix degradation re- quires the existence of specific metalloproteinases. To date three metalloproteinases, collagenase, gelatinase, and stro- melysin, have been isolated that exhibit matrix degrading activity (4). Collagenase cleaves collagen types I, 11, and 111 at a single locus and collagen X a t two (4, 5). Gelatinase has activity against denatured collagen (gelatin) and collagen types IV and V (6-10). Stromelysin, with the widest substrate

* This work was supported by Grants AR 33714 and GM 34090 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “eduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence($ reported in this paper has been submitted to the GenBankTM/EMBt Data Bank with accession numberrs) J04 732.

$ Profesor Titular en Comision de Servicio of the University of Valencia, Spain. Present address: Dept. of Biochemistry, Faculty of Pharmacy, University of Valencia, Valencia 46010, Spain.

§ Present address: Dept. of Medicine, Stanford University School of Medicine, Stanford, CA 94305.

specificity of all, can degrade fibronectin, laminin, collagen IV, and cartilage proteoglycans (11). These three enzymes exhibit complementary, but little or no common catalytic function. However, comparison of cDNA-derived amino acid sequences for these proteinases indicate that they share a high degree of homology in primary and secondary structure

Precisely how the production of these metalloproteinases is regulated is important in understanding the physiologic and pathologic processes involving extracellular matrix. In human and rabblt fibroblast cultures, collagenase and stromelysin production is coordinately regulated in response to several stimuli. Enzyme synthesis is induced by a number of factors that include interleukin-1P and the tumor promoter TPA’ (12-0-tetradecanoylphorbol-13-acetate). Induced levels can be subsequently suppressed by retinoic acid or dexamethasone (14-17).

Transin (18), the rat homologue of stromelysin, is induced in fibroblasts transformed with polyoma virus, Rous sarcoma virus, or oncogene H-ras and in fibroblasts exposed to epider- mal growth factor. The induction of transin transcription by epidermal growth factor can be blocked or suppressed by prior or subsequent exposure of the cells to transforming growth factor p (19).

Recently, we have isolated cDNA clones that cover the complete coding region for human stromelysin and have used them to establish that IL-lp, retinoic acid, and dexametha- sone modulate the accumulation of stromelysin mRNA (14). We have since isolated genomic clones for human stromelysin. In this report we demonstrate by transient gene expression assay that 1.3 kb of the stromelysin promoter region contains DNA elements responsible for IL-lP induction and dexameth- asone suppression. Induction by IL-lP is decreased from approximately 14.4-fold with the 1.3-kb promoter fragment to approximately 7.6- and 6.5-fold with the 0.74- and 0.47-kb fragments, respectively, thereby suggesting that the strome- lysin promoter contains multiple IL-lp-responsive elements. With all three promoter constructs, dexamethasone sup- pressed the inductive capability of IL-lp by approximately 50%.

(12-14).

MATERIALS AND METHODS

Chemicals and Enzymes- Klenow enzyme, restriction endonucle- ases, and calf thymus DNA were from New England Biolabs (Beverly, MA) and Boehringer Mannheim (Indianapolis, IN). Bluescribe and Bluescript vectors and TS and T, polymerases were from Stratagene (La Jolla, Ca). MI3 vector and linkers were obtained from Pharmacia LKB Biotechnology, Inc. (Piscataway, NJ), and the human growth hormone vectors came from Nichols Institute (San Juan Capistrano,

’ The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13- acetate; IL-lp, interleukin-lp; kb, kilobase pair; bp. base pair; hGH, human growth hormone; SDS, sodium dodecyl sulfate; Hepes, N-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid.

8339

8340 Regulation of Human Stromelysin Gene

C.4). [a-”PIUTP (800 Ci/mmol), [35S]dATP (1200 Ci/mmol), and [y3’P]ATP (6000 Ci/mmol) were from Du Pont-New England Nu- clear (Wilmington, DE). Human recombinant IL-10 was obtained from either Cistron (Pine Brook, NJ) or Boehringer Mannheim.

Isolation of Stromelysin Genomic Clones-A human stromelysin cDNA (14) was cloned into Bluescript KS (+)-vector and used to make RNA probes to screen a human “X fix” genomic library (Stra- tagene). The plasmid DNA was restricted at the 3’-end of the coding region so that only 3ZP-labeled transcripts originating at the 5’-end were synthesized. The filters were hybridized in 5 X SSPE (1 X SSPE, 150 mM NaCl, 10 mM NaH2P04, 1 mM EDTA), 0.1% SDS at 68 “C overnight and then washed in 0.1 X SSC (1 X SSC, 150 mM NaC1,15 mM Na-citrate), 0.1% SDS at 75 “C. Of the approximately 5 X lo5 plaques screened, 10 signals were recovered and 5 of those were purified. One clone (HAF10) that was characterized by restriction mapping and partial nucleotide sequencing is 14 kb in length and contains approximately 10 kb of sequence upstream of the transcrip- tion start site.

Nucleotide Sequence Analysis-Restriction enzyme fragments of HAFlO were cloned into M13 vectors (20) and sequenced according to the dideoxynucleotide chain termination method (21) using [35S] dATP (22), modified T7 DNA polymerase (23), and the “sequenase” kit from United States Biochemicals Co. (Cleveland, OH). Sequence specific oligonucleotides (17-mers) used as primers were made on an automated oligonucleotide synthesizer (Biosearch, Inc., New Bruns- wick, NJ). Each strand was sequenced at least twice.

Transcription Start Site-A 40-mer oligonucleotide (24 ng) com- plementary to stromelysin mRNA sequence (nucleotides 66-105 of the protein coding region) was end-labeled by [Y-~’P]ATP and TI polynucleotide kinase and hybridized with total RNA (30 pg) from induced fibroblasts in 50 pl of 80% formamide, 0.4 M NaCl, 10 mM Hepes, pH 7.5, at 30 “C overnight. After ethanol precipitation, the reverse transcriptase reaction was carried out as described (24, 25). For accurate determination of the transcription start site, the primer-

CONTROL IL-1

STROMELYSIN r*

fl-ACTIN

FIG. 1. Transcriptional activation of stromelysin after stimulation with IL-la. Nuclei were isolated from cultured human skin fibroblasts after induction with IL-1P (5 units/ml) for 3 h. Initiated transcripts were labeled as described under “Materials and Methods” and hybridized to stromelysin and &actin cDNAs (5 pg of plasmid DNA/slot).

FIG. 2. Partial restriction map of HAFlO and promoter f ragments used in the transient gene expres- sion constructs. At the top of the fig- ure is a schematic of the X genomic clone HAF10. The positions of the SstI ( S ) and XbaI (X) sites are drawn to scale. Directly below is an enlargement of the SstI partial XbaI 2.1-kb fragment con- taining 1.3 kb of promoter sequence ex- tending through the first exon (box) and into the second (half-box). The restric- tion sites used to obtain the transient gene expression constructs are noted. In the constructs the promoter elements used to drive the hGH gene were derived from the SstI-AuaI (1293 nucleotides; -1303 to -ll), Asp718-AuaI (744; -754

and Sau3A-AuaI (43; -53 to -11) frag- ments.

to -ll), X~UI-AUUI (468; -478 to - l l ) ,

1 kb - S

extended product was run on a sequencing gel along with the sequenc- ing reactions of the 1179-bp XbaI-XbaI fragment containing the first exon using the same 40-mer as primer.

Plasmid Construction-A 2.1-kb fragment of HAF10, containing 1.3 kb of promoter sequence and extending into the second exon of stromelysin, was isolated by SstI and partial XbaI digestion and cloned into Bluescribe. This plasmid was digested with AuaI which cuts at -11 respective to the transcription start site. The ends were filled in with Klenow enzyme and BamHI linkers were attached to replace the AuaI site. Subsequently, using HindIII linkers, the SstI site of this plasmid was converted to a HindIII site. The resultant plasmid was designated as B8.218. To obtain the 1293-bp promoter construct (-1303 to -ll), the HindIII-BamHI fragment of B8.218 was cloned into the human growth hormone reporter vector p$GH (26). To make the 744-bp construct, the 1293-bp construct was digested with HindIII and Asp718 to remove 560 nucleotides on the 5’-end of the promoter, followed by blunt-ending and religation. The 468-bp construct was obtained by cloning the corresponding XbaI- BamHI fragment of B8.218 into p$GH. A minimal construct contain- ing only 43 nucleotides of promoter sequence (-53 to -11) including the TATA box was obtained by cloning the corresponding Sau3A- BamHI fragment of B8.218 into BamHI-digested p$GH. Plasmid constructs containing the correct orientation of this promoter frag- ment were identified by sequencing.

Transfections and Gene Expression Assays-Human fibroblasts prepared from explant of infant foreskin by standard techniques were cultured and maintained as described (14). For the transfection experiments, cells between 5 and 15 passages were seeded at a density of 7 X lo5 cells/lOO-mm dish and maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and antibiotics. The next day the media were changed and 4 h later the cells were transfected with 40 pg of total DNA (20 pg of reporter and 20 pg of carrier calf thymus) in calcium phosphate (27) for 16 h. At 24 h post- transfection, the cells were treated with IL-1P (5 units/ml) for an additional 48 h and the secreted hGH levels were then measured using a solid-phase radioimmunoassay kit (Nichols Institute). In other experiments, the cells were induced for 3 h with IL-1P, washed 3 times with Hanks’ balanced salt solution, and the media replaced along with the addition of M dexamethasone. Forty-eight hours later the media were assayed for hGH. To provide an internal control for DNA uptake, some experiments included co-transfection with the RSVcat vector (28). Chloramphenicol acetyltransferase assays were performed on the cell extracts as described (28). We detected no measurable plate-to-plate or treatment-derived variation in chlor- amphenicol acetyltransferase activity.

Nuclear Run-on Assay-Nuclei were isolated by a modification of the methods as described (29, 30). For each experiment, 2.5 X lo7 nuclei were incubated in a reaction mixture containing 30 mM Tris- HCl, pH 8.0, 5 mM MgClz, 150 mM KCl, 2.5 mM dithiothreitol, 100 units/ml RNasin, 1 mM ATP, GTP, and CTP, and 200 pCi [a-”P] UTP for 30 min at 30 “C. After DNase and proteinase K treatment the labeled transcripts were hybridized at a concentration of 7 X lo6

S I I

I

X

Sstl Asp718 Xbal

U Sau3A

hQH

Regulation of Human Stromelysin Gene 8341

cpm/ml to cDNA bound onto a nitrocellulose filter in 5 X SSPE, RESULTS AND DISCUSSION 0.1% SDS at 68 "C for 48 h. The filters were washed at 68 "C in 0.1 1 ~ - 1 p promotes the accumulation of stromelysin m~~~ in x SSC, 0.1% SDS. cultured human fibroblasts and, conversely, retinoic acid or a dot matrix program written by T. Burglin, Massachusetts General dexamethasone suppress the induced levels (14). In Order to Hospital, and subsequently aligned using the multiple sequence editor determine whether the IL-lp modulation of stromelysin gene written by W. Gilbert, Whitaker College. expression reflects changes in promoter activity, we per-

Promoter Sequence Comparisons-Sequences were compared using

Sst I GAGCTCTGGGATCMGTGATTCTCCTGCCTCAACCTCTCAAAGTGCTAGGATTACAGACATGGGTCACGGCACCTGGCCTAAAGACATTTTAAMCTAGTATTCTATGGTTCTCCATTCC -

- 1300 - 1280 - 1260 - 1240 - 1220 - 1200

TTTGATGGGGGGAAAAMCCATGTCTTGTCCTGATTG~TACAGG~TATTTGGCCACATTGATATGAGGACMGGA~CAGAGTGGTGGCAGTGATGTGMTTCCAGGMGAM

-1180 -1160 -1140 - 1120 -1100 - 1080

CAGTGGAGCCCMCAGATAAAATATTGAGAGGTTAAAAGTGTAGMGCTGTGGCAGCTCTMTTCCTTCTATATTMGGTTTATCCTTTGTAMTTTTCTCTGGAGTTATMCTTATAM

- 1060 - 1040 - 1020 - 1000 - 980 - 960

ACATATTTAAAGTTTAGAGGAAAGCCTGATAGATAATATGTTTGCTTTTCCMCTGTTMGATGTGATTGGCAAMTGGGCCATGCTGTTCCT~CTAMTACAAMGTTTGCTATATT

- 940 -920 -900 -880 -860 -840

Asp718 TTAAMCAGCTTCGACGACTTCCCTGGAAGATGTTTCCCAAGTCCCAGGMGAGTCATTMTCATTGTTCMT~TTGTACCTGCCTCTGCTACTCTATTTTCCACACTGMCTGT

-820 - 800 - 780 - 760 - 740 - 720

AGTCAMTGGTTTATCTGTCTCTCTCATTCTAATGTMGCTACTTGAGGGTATGGACCATGCCTCATTTGATTCTGTATTCCTAGTACTTAGCATGGTATCTGATATATAGTAGGTMCC

- 700 -680 -660 -640 - 620 -600

XbaI AAMATTACTTAGAGMCTAAATAATATATTAGTTATGAGTGAMGGCTCATAGCAMTGTCAMCAGGTATTAAMGTC~TCCAAMATCATGCAGACATTTCTAGATATTA

- 580 -560 . -540 -520 -500 - 480

Sau3A Ava I 7 TGGAMTGGTCCTGCTGCCATTTGGATGAAAGCAAGG~~~CTGCGGGT~TCCAMCAMCACTGTCACTCTTTAAMGCTGCGCTCCCGAGGTTGGACCTACMGGAGGCAG - -

-100 - 80 -60 -40 - 20 1

Met GCMGACAGCMGGCATAGAGACAACATAGAGCTAAGTAMGCCAGTGCAMTG -

20 40 60

FIG. 3. Nucleotide sequence of the first 1.3 kb of the human stromelysin promoter. The presented sequence extends 5' from the first ATG-codon (Met) through the transcription start site (arrow) to an SstI site (underlined) 1.3 kb further upstream. All the restriction sites utilized for making the transient gene expression constructs are marked. The TATA box is located between positions -30 and -24. The boxed sequence centering around position -65 shares strong homology (87.5%) with the TPA responsive element (36). Also underlined are three sequences that share strong homology with the consensus sequence for the glucocorticoid-responsive element (33).

8342 Regulation of Human Stromelysin Gene

formed nuclear run-on assays. Nuclei from IL-I/?-treated hu- man fibroblast cultures contained increased amounts of stro- melysin transcripts when compared to nuclei from control cells (Fig. 1). By densitometric scanning, the difference in intensity of these bands is about %fold (2.9 & 1.1). In contrast, IL-10 treatment did not change the amount of radioactivity in P-actin transcripts, suggesting that IL-lP does not have a generalized effect on transcription. Based on these results, the modulated expression of stromelysin can be at least par- tially attributed to changes in promoter activity.

In order to further study the promoter regulation of human stromelysin, we isolated and characterized a 14-kb genomic clone, HAFlO (Fig. 2). A 2.1-kb region, containing 1.3 kb of promoter sequence and extending into the second exon of stromelysin, was characterized by nucleotide sequencing. In Fig. 3 the nucleotide sequence of the 1.3-kb promoter and the 5”untranslated region is presented. The transcription start site was determined by primer extension of a 40-mer oligo- nucleotide complementary to a segment of the first exon using total RNA from induced fibroblasts as template (see “Mate- rials and Methods”). One major product was detected as well as faint products one nucleotide above and one and two nucleotides below (Fig. 4). When similar analysis was carried out with total RNA from control cells, which make only small amounts of stromelysin (14), the primer-extended product was barely visible (data not shown).

For the transient gene expression assays, restriction frag- ments of stromelysin promoter were cloned into the hGH reporter vector (26). After transfection of human fibroblast cultures, one set of cultures was treated with interleukin-1P. After 48 h (72 h post-transfection) the amount of hGH secreted into the culture media was determined by radio- immunoassay (Table I). With the 1293-bp promoter con- struct, IL-l/? induced the accumulation of 14.4-fold more hGH

PE

/ \ T - A*

I

3’

FIG. 4. Mapping of the transcription start site of human stromelysin. Single lane at left ( P E ) is the product of the primer extension of a 40-mer oligonucleotide using induced total RNA as template (see “Materials and Methods”). The four right lanes are the sequence of a stromelysin genomic fragment using the same oligo- nucleotide as primer (order of lanes from left to right is G, A, T, and C). The sequence 5’-GACCTACAAGG-3’ is the complement of that given by the sequence reactions. The arrow and asterisks denote the positions in the sequencing gel and in the corresponding complemen- tary sequence, respectively, of the major primer-extension product.

TABLE I IL-10 and dexamethasone modulation of stromelysin promoter

activity as evidenced by transient gene expression assay A, 24 h post-transfection, IL-1p was added to the media. Secreted

hGH was assayed 72 h post-transfection (48 h after addition of IL- I@). IL-lp-induction denotes the ratio of hGH in media of IL-10- induced cells to that of transfected cells incubated for same time without IL-1p. The control plasmid was pXGH5. B, 24 h post- transfection, the cultures were stimulated for 3 h with IL-16, the media changed, and dexamethasone added along with fresh media (as in Ref. 14). Secreted hGH was assayed 75 h post-transfection (48 h after addition of dexamethasone). IL-lp induction was calculated as in A. The control plasmid was pTKGH. Each value is the average of a t least four experiments with the exception of the dexamethasone studies using the 744, 468-, and 43-bp constructs which are the average of two.

A B Stromelysin

promoter IL-1p induction IL-10 induction Dexamethasone (48 h) (3 h) suppression

bp % Control 1.2 1.2 0

1293 14.4 6.5 57 744 7.6 3.7 62 468 6.5 3.4 50

43 1.0 1.0 0

as compared to untreated cultures. As the length of the promoter fragment attached to the hGH gene was reduced, the IL-lP inducibility of the promoter diminished. Using the 744- and 468-bp constructs, IL-1/? induction dropped off to 7.6- and 6.5-fold, respectively. Finally, with the 43-bp minimal promoter construct (-53 to -11) containing only the TATA box, there was no measurable response to IL-l/? treatment. Endogenous hGH gene activity was not detectably affected by IL-l/? treatment as determined in mock-transfections of carrier DNA alone nor was the control plasmid driven by the metallothionein promoter (pXGH5) induced by IL-1P. Based on these results, we suggest that there are at least two DNA elements in the human stromelysin promoter that are respon- sive to the IL-1/? induction, one within the 744-bp fragment and another further upstream in the 1293-bp fragment.

We have also studied the suppression of IL-lfl-induced stromelysin expression by dexamethasone. After transfection, cell cultures were treated with IL-l/? for 3 h followed by the addition of dexamethasone. After a further incubation of 48 h, hGH was quantitated in the culture medium (Table I). With all the promoter constructs tested, dexamethasone sup- pressed the IL-1/? inducibility by about 50%. The hGH coding region contains a sequence which is positively regulated by glucocorticoids in some cell types (26). However, in our ex- periments dexamethasone treatment had no significant effect on hGH levels in mock-transfected cultures nor in cultures transfected with control plasmid pTKGH (thymidine kinase promoter attached to hGH gene) (data not shown). These data indicate that the 1.3-kb stromelysin promoter contains DNA element(s) involved in suppression by dexamethasone.

Dexamethasone is known to both stimulate and, in some cases, suppress gene expression through the binding of a receptor-hormone complex (31, 32) to the same consensus DNA sequence GGTACANNNTGTYCT (33). When we looked for such a consensus sequence in the human strome- lysin promoter we could find three sequences (-1300 to -1286, -871 to -857, and -150 to -136) which had 2 mis- matches on the 5’-end sequence and 1 or 2 mismatches on the 3’-end sequence (see Fig. 3).

Comparison of the stromelysin promoter sequences from human, rabbit (34), and rat (35) reveal a very high degree of sequence similarity between the promoters (Fig. 5). For ex-

Regulation of Human Stromelysin Gene a343

CATGAGGTAGGAGTGAGTTGGTGAGTGGGAGAGCATCCTCATAGMCCAGGGGAGGGAGGACTGGATAGGGGGTTTGCAAAGGGMAACCACAAMGCGGATMCATTTGAAATGTAAAA

I I I I I I I

I I I I I I I AGCTTGATATCCMTTCCTGCAGCCACTTATGGAGMGATATTTGGAGTCTATCMCAAAATATTMGCMCCAAGMCACACGATTTCTCCTTTCTACAGACCCTCGAT

AGTCAMTGGTTTATCTGTCTCTCTCATTCTMTGTMGCTACTTGAGGGTATGGACCATGCCTCATTTGATTCTGTATTCCTAGTACTTAGCATGGTATCTGATATATAGTAGGTMCC .....I ......... I ......... I ......... I ......... I ......... I ......... I ......... I ......... I ......... 1 .........I ......... I ....

- 740 - 730 - 720 -710 - 700 -690 -680 - 670 -660 -650 -640 -630

AAMAAMAAMTMCCMTAAAAMTATCATAAAATMGAAAGACAAATCTGACAG~GATATMGATAAAACCAGGCCATTCTACTTCAGTAMTCATATCMTTATATGAGCCTT

II I I I I I II I I I

II I I I I I II I II CTGTTCTTGTCCATGAGACGTTCCTCCTCMGGACTMCAAATCCCACAGAAATCACTMTAAATATATCAGATCACAGACTTAGAAAACGMCTCTCCTTCCAMTTTTCTTTAGATAC

MAMTTACTTAGAGMCTAMTMTATATTAGTTATGAGTGAAAGGCTCATAGCAMTGTCAMCAGGTMATTMMGTCAAAAAATCCAAAAATCATGCAGACATTTCT--AGATAT

.....I ......... I ......... I ......... I ......... I ......... I ......... I ......... I ......... I ....... :.I ......... I ......... I .... - 620 -610 - 600 - 590 - 580 -570 - 560 -550 -540 -530 -520 -510

TATAGAAAAMCTATTATAGACCCATCTCCTTTTMTATATGGGGTACAGMTGTGGGTCGTAGTGACAGACAGCGTGGGCTGTGGCTACAGAGGCACACCTTGTCCTTACCTCATCTCA

II I I I I I I I I I I II I I I II I I IIII

II I I I I I I I I I I I I I l l

.....I ......... I ......... I ......... I ......... I ......... I ......... I ......... I ......... I ......... I ......... I ......... I ....

CATTTCCTGTATTTTAGAGTATMTMTG-------CAGGCTCTAGCTATTCACGTGCAGTTT---------- - - TTCATTTGCTTAGATTTTGGTGCATTTMTTCTTAAACATGTTCT

TATCTTTTATGTTTT-GAGTATMTTGTATATAGTATAGACTATAGCTATGTATGTACACTTTCCACTTACATCTTTTATTTGCTT--- - - - - - - - - - - - - - - - - - - TTATA-ATGTCTT

- 500 - 490 - 480 - 470 - 460 -450 -440 -430 -420 -410 - 400 - 390

CTGTATTCAGCTTTGACTTCTG~GTTCTTTGTACMTTTGGACTTTTTACCMGTTAGGCCACTACT---ATCCMGTCATAAACATTACAGCTTCT---GMGGATAGTTACATTTT

II II I IIIIIII I IIIIII IIIIIIIIIIII II I I I l l II I I I I I I I I I I IIII I I I l l I IIII

II II I IIIIIII I IIIIII IIIIIIIIIIII II I I I l l I I I I I I I I I IIIII I I I I I I I II I l l I I I I I TCCTMMTAMGCCACTCTTMMGTTCT--GCACMTTCTGACTTTTTACCAGGTCAACC---TTCTTCTCCCAMCACATGMCATMGTTTTTCTAGTAAATTCTAGTCAMTTTT

TCTTMMTMMCTGCTTTTAGMGTTCT- -GCACMTTCTGA-TTTTTACCMGTCMCCTACTTCTTCTCTCMMGGACAAACATAMTTGT-CTAGTGMTTCCAGTCMTTTTT ......... ......... ......... ......... ......... ......... .... .....I ......... I ......... I ......... I ......... I ......... I I I I I I I

- 380 - 370 -360 -350 - 340 -330 -320 -310 -300 - 290 - 280 - 270

C C A M - G T A G M - - - - - A M M T G C C C C A G T T T T C T C T T T T G C T M G G C A G G M G C A T T T C C T G G A G A T T M T C A C C A T - T C G C T T T G C A A M T T M G M G G T T T G A A ~ C T ~ G T A M

I l l I I I II IIIII II IIIIIIIIIII I I I II IIIIIIIII IIIIIIIIIIIIIIIIII I I II II I I I I I I I I I I I I I I IIII I I IIII

I l l I I I II I l l II II l l l I IIIII I I I II I l l l I I I I I I I I I I I I I I I I I I I I I I I I l l II II I I I I I I I IIIIIII I I I I I I IIII

.....I ......... I ......... I ......... I ......... I ......... I ......... I I I I I I

CCAGAGGTMAAMMMMAAAGCCCCAGTTTTCTCTTTTGCCMCACAGGMGCATTTCCTGGAGATTMTCACTGTGTTGCCTTACAAMTTGGGAAGGTTGAGAGAMTTACTAM

C C A G M G A A A " - - - - - - AMTGCTCCAGTTTTCTCCTCTACCMGACAGGMGCACTTCCTGGAGATTAATCACTGTGTTGCCTTGCMMTTGGGAAGGTTGAGAGAMTTAGTAGTAM ......... ......... ......... ......... ......... ....

- 260 - 250 -240 - 230 - 220 -210 - 200 j9+I - 180 -170 -160 [ GMGATTATATCACTCTTC--TGATTTTT--MTTTTTGGAMTGGTCC------ CATTTGGATGGAAGCMTT GAGTCA TTGCGGGTG-ACTCTGCAMTACTGCCACT A T M

I II II IIIII II I I l l I l l I l l IIIIIIIIIIIIII I I IIIIII I I I I I I I I I I I I I I l l I II I I l l1 I I II II I l l

I II II IIIII II IIIIII I l l I l l IIIIIIIIIIIIIIII I I I I I I I I I I I IIII IIIIIIII I l l I II I I I I I I I I I II I l l

.....I ......... I ......... I ......... I ......... I ......... I ......... I I I I I I

GTAGATTGTATCATCCTACTTTGAGTTTAAAAATCTTTGGMATGGTCCTGGTGTCGTATGGATGAAGGCA--- GAGTC CTGCAGATGTACTMACAAACATTCCTACC A T M

GTAGGTTGTATCATCCTACTTTCMTTTGG-MTGTTTGGAMTGGTCCTGCTGCCATTTGGATGAAAGCMG GAGTCA CTGCGGGTGATCCAAACAMCACTGTCACT T T M ......... ......... ......... ......... ......... ....

- 140 - 130 -120 -110 - 100 - 90 - 80 - 70 -60 - 50 -40 - 30

3 TTGGGCTCAGAMGGTGGACCTC

I l l II IIII I I II I

I l l II IIII I I I I I

.....I ......... I ......... I

TTGAGCTCTGGAGAGTAGATGTA

CTGCGCTCCCGAGGTTGGACCTA

- 20 -10 +1

FIG. 5. Nucleotide sequence comparison of the stromelysin promoters of rat (top l ine), rabbit (middle line), and human (bottom line). The lines between the sequences of each species denote nucleotide residues shared by all three promoters. The mark +I is the transcription start site for human stromelysin. The TPA responsive element and the TATA boxes are boxed.

a344 Regulation of Human Stromelysin Gene

ample, the first upstream 370 nucleotides of human and rabbit stromelysin promoter are 82% identical in sequence. Further- more, between all three promoters there is a 69% sequence homology in the same region. The high degree of nucleotide sequence similarity suggests that this region is important for stromelysin regulation. In all species examined TPA and IL- I@ induce stromelysin production, and retinoic acid and dex- amethasone suppress it (14-17, 35). TPA exerts its effects at the level of transcription (15, 35, 36). The DNA element responsive to TPA has been identified in the promoter of human collagenase and the homologous sequence has been found in the human metallothionein and rat stromelysin promoters (36, 37). The TPA-responsive element of the rat stromelysin promoter falls within the region of strong homol- ogy discussed above (see Fig. 5) and shares all but one nucleo- tide with its rabbit and human counterpart. It is, therefore, very likely that the same regions of the rabbit and human promoters are involved in the TPA response.

Previously, a 700-bp promoter fragment of rabbit strome- lysin was shown to be responsive to induction by IL-1P, epidermal growth factor, TPA, or CAMP and to subsequent suppression by dexamethasone (34). By nuclear run-on and transient gene expression assay we show that human stro- melysin is induced by IL-l@ at the level of transcription and that the 1.3-kb promoter fragment contains IL-l@- and dex- amethasone-responsive DNA elements. In fact, the human promoter appears to contain at least two DNA elements responsive to IL-1P, one of which is located a minimum of 754 by upstream of the transcription start site. It would be interesting to determine whether rabbit and human strome- lysin share the same distribution of IL-l@-responsive ele- ments.

Acknowledgments-We thank Larry Kedes for the human @-actin cDNA, Thomas Burglin for the promoter sequence comparisons, and Francine Mittleman for help in the preparation of this manuscript.

1. 2.

3.

4.

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