THE JOI~NAL OF Vol. 269, No. 14, Issue of April 8, pp ... · THE JOI~NAL OF BIOLOGICAL CHEMISTRY...

THE J O I ~ N A L OF BIOLOGICAL CHEMISTRY Vol. 269, No. 14, Issue of April 8, pp. 10363-10369, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Phenotypic Alterations in Fos-transgenic Mice Correlate with Changes in Fos/Jun-dependent Collagenase Type I Expression REGULATION OF MOUSE METALLOPROTEINASES BY CARCINOGENS, TUMOR PROMOTERS, CAMP, AND FOS ONCOPROTEIN*

(Received for publication, December 20, 1993)

Sabine GackS, Riidiger VallonS, Jorg SchaperS, Ulrich RiitherQ, and Peter Angelh From the $Kernforschungszentrum Karlsruhe, Znstitut fur Genetik, Postfach 3640, 76021 Karlsruhe, Federal Republic of Germany and the PMedizinische Hochschule Hannouer, Znstitut fur Molekularbiologie, 30623 Hannover, Federal Republic of Germany

Using specific cDNAs isolated from mouse fibroblasts we determined tissue-specific expression of different matrix metalloproteinase genes: both stromelysin-1 and collagenase IV are highly expressed in heart and lung, whereas collagenase I is expressed most abundantly in skeletal muscle, kidney, and bone. High basal level expression of stromelysin-2 is found in heart and kidney. Like in man and rat, the expressions of collagenase I, stromelysin-1, and stromelysin-2 are regulated by the tumor promoter 12-0-tetradecanoyl-phorbol 13-acetate and by W irradiation, but not by cAMF? In contrast, the expression of the 72-kDa collagenase IV is not affected by either stimuli. We and others have shown previously that under cell culture conditions, the regulation of human collagenase I is regulated by the transcription factor FodJun (AP-1). Here we show that in c-foe transgenic mice transcription of collagenase I is induced in thymus, spleen, and, most dominantly, in bone upon overexpression of Fos. Neither collagenase IV nor stromelysin-1 or stromelysin-2 expression is affected by c-Fos. The sites of induced collagenase I expression correlate with the sites of Fos-induced long-term cellular alterations in transgenic mice including bone remodeling and T cell development. In fact, in the developing bone tumors strongly enhanced levels of collagenase I transcripts were detectable. These results identify collagenase I as a Fos-regulated gene in vivo and suggest a possible role for FodJun heterodimers in establishing the pathological phenotype of c-fos transgenic mice.

Resident cells of tissues are capable of secreting an array of structurally related zinc endopeptidases known as matrix metalloproteinases (MMPs).’ The MMPs initiate the degradation of the surrounding macromolecules of the extracellular matrix (ECM), mostly proteoglycans and the different specific types of collagen. ECM degradation presumably contributes to the ini- tial phase of tissue remodeling inherent to the physiological processes of morphogenesis, angiogenesis, involution of the uterus, bone resorption, inflammation, and wound healing (for

* This work was supported by a grant from the Deutsche Forsch- ungsgemeinschaft (An 182/6-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must

U.S.C. Section 1734 solely to indicate this fact. therefore be hereby marked “advertisement” in accordance with 18

ll To whom correspondence should be addressed: Kernforschungszen- trum Karlsruhe, Institut f i r Genetik, Postfach 3640, 76021 Karlsruhe, Germany, Tel.: 07247-823-444; Fax: 07247-823-354.

The abbreviations used are: MMP, matrix metalloproteinase; ECM, extracellular matrix; TPA, 12-0-tetradecanoylphorbol-13-acetate; PCR, polymerase chain reaction; kb, kilobase paids); TIMP, tissue inhibitor of metalloproteinases.

review, see Refs. 1-5). Aberrant activity of MMPs has been found to be involved in a variety of pathological processes such as rheumatoid joint destruction, corneal ulceration, metastasis of tumor cells, and genetic diseases (e.g. recessive dystrophic epidermolysis bullosa; for review, see Refs. 3-6).

The various members of the MMP family contain several distinct domains that are highly conserved. Despite this high degree of similarity, the MMPs identified so far differ in substrate specificity and expression in response to extracellular stimuli, suggesting that each individual member of the MMP family has a distinct function in the physiological and pathological processes listed above (1-3, 6). Furthermore, the activity of these enzymes is tightly regulated on several levels: regulation of transcription (7-151, activation of the latent proenzyme (16-191, and interaction with specific inhibitors of MMPs (TIMP-1 and TIMP-2, Refs. 20-22). Studies in cell culture systems have revealed multiple mechanisms that posi- tively or negatively interfere with the transcription of MMPs. Transcription is enhanced by carcinogens, cytokines, and tumor promoters (for review, see Refs. 4 and 23); and repressed by steroid hormones (24-26) and by the E1A products of adenovi- rus (25, 27, 28). The transcription of human collagenase I and of human and rat stromelysin-1 are regulated by the transcription factor AP-1 in most of the cell systems examined so far (10, 29-32). AP-1 is a heterodimeric complex whose subunits are encoded by members of the j u n , fos, and ATF gene families (for review, see Ref. 23). AP-1 activity is repressed, presumably by physical interaction with the activated steroid hormone recep- tors (26, 33).

Despite the great body of information of MMP expression in cultured cells, little is known about the expression and function of distinct MMPs in the intact multicellular organism. For example, expression of collagenase type I has been found to be regulated by basic fibroblast growth factor and transforming growth factor p (12, 341, both of which play a decisive role during early development (35). In fact, changes in the expression and activity of MMPs have been observed during in vitro differentiation of mouse F9 and PSA-1 cells, and during peri- implantation development of mouse embryos (36). Spatial and cell type-specific expression in adult mice or during mouse development (e.g. by in situ hybridization) has been examined only for the 72-kDa form of collagenase type IV (37).

To be able to dissect the complex regulation of MMP gene expression in mouse embryos and adult mice, and to determine the mechanisms of transcriptional regulation of mouse MMP expression, we isolated specific mouse probes encoding MMP-1 (collagenase type I), MMP-2 (72-kDa collagenase type IV), MMP-3 (stromelysin-l), and MMP-10 (stromelysin-2). These clones could be unequivocally assigned by their homologies to rat and man. High degrees of tissue-specific expression support

10363

10364 Fos-dependent Expression of Matrix Metalloproteinases in Adult Mice

that the MMPs have defined functions. These differences in tissue-specific expression in the intact organism may be explained by differences in gene regulation observed in mouse fibroblasts in response to extracellular signals including the tumor promoter TPA, CAMP, and UV irradiation. In light of the essential role of J u f l o s (AP-1) in the regulation of human collagenase I (at least under cell culture conditions), we found strongly enhanced expression of collagenase I in liver, spleen, thymus and, most dominantly, in bone of c-fos transgenic mice. In contrast, neither collagenase IV nor stromelysin-1 and stromelysin-2 expression are affected by Fos. The tissue-specific induction of Fos-dependent collagenase I expression correlates with the sites of Fos-induced phenotypic alterations including disturbances of bone remodeling and T cell development in the thymus and enlargement of the spleen (3840). Depending on the type of expression vector in transgenic mice, long-term overexpression of Fos results in the formation of bone tumors (40). Such tumors were found to express strongly enhanced levels of collagenase I. In other tissues expressing enhanced levels of exogenous c-Fos, neither tumor formation nor enhanced levels of collagenase transcripts were detectable. These results identify collagenase type I as the first example of a Fos-regulated gene in animals and suggest a decisive role of the FosIJun-dependent program of gene expression, including collagenase I, in establishing the pathological phenotype of transgenic animals overexpressing Fos and possibly other on- cogenes acting through Fos.

MATERIALS AND METHODS Cell Culture-NIH 3T3 cells, SV40 transformed mouse lung fibro-

blasts (LuSVX), and ras-transformed rat fibroblasts (rat2pEJ)' were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. F9 mouse teratocarcinoma cells were cultured in Dul- becco's modified Eagle's mediud-12 (l:l), 10% fetal calf serum, 2 m~ L-glutamin, 170 JIM /3-mercaptoethanol (41). As indicated in the figure legends, cells were treated with CAMP (8-bromoadenosine-3',5'-mono- phosphate) and TPA at a final concentration of 1 m~ and 60 ng/ml, respectively. For W treatment, the tissue culture medium was removed and the cells were washed twice with phosphate-buffered saline (37 "C). After W irradiation (30 J/m'), the original culture medium was put back on the cells (34,421. After 24,36, or 48 h, cells were lysed and RNA was prepared. Prior to the various treatments (except for W), cells were synchronized in the GdG, phase of the cell cycle by culturing the cells in serum-free medium for 24 h.

Analysis of Gene Expression-RNA from tissue culture cells was prepared by lysing the cells in 100 m~ NaCl, 20 m~ Tris-HC1, pH 7.4,lO m~ EDTA, 0.1% SDS, and proteinase K (300 pg/mlf. The cell lysate was incubated overnight with oligo(dT)-cellulose type VI1 (Pharmacia). Af- ter extensive washing with HSB (300 m~ NaCl, 10 rn Tris, pH 7.5, 5 m~ EDTA, 0.1% SDS; to remove DNA and nonpolyadenylated RNA), poly(A+) RNA was eluted with water and concentrated by ethanol pre- cipitation. Preparation of total RNA from tissues of mice was described previously (38). 20 pg of total RNA from mice or 10 pg of poly(A+) RNA from tissue culture cells were separated on a 1% agarose gel in 10 m~ NaH,P04/Na,HP04, pH 6.85, and transferred to a nylon membrane (Hybond N', Amersham). The membrane was prehybridized in 600 m~ NaC1, 60 m~ sodium citrate, pH 6.5 (4 x SSC), bovine serum albumin, Ficoll, and polyvinylpyrrolidone, 0.02% each, and 3.3% P,PPi (0.2 M NaH,PO,, 0.3 M N+HPO,, 1.5% Na,P,O,), and 20 pghl salmon sperm DNA at 65 "C. Hybridization was performed in prehybridization solution containing the radioactively labeled probe (40 n g h l final concentration) at 65 "C for 16 h. Non-bound probe was removed by washing the filter at 65 "C for 30 min in 300 m~ NaC1,30 m~ sodium citrate, pH 6.5 (2 x SSC), 1 x SSC (twice), and 0.5 x SSC (each washing solution containing 0.1% SDS, 3.3% P,PP,). Radioactively labeled probes were prepared by PCR (1 min 94 "C, 1 min 60 "C, 2 min 72 "C; 35 cycles) using the sequence-specific primers described below and purified cDNA fragments as templates. Analysis of tissue-specific expression of MMPs in adult mice was done using a multiple tissue Northern (MTN, Clon- tech Laboratories), containing 2 pg of highly purified poly(A+) RNAfrom different tissues of mice as described in the figure legend.

M. Giles, H. Ponta, and P. Herrlich, unpublished data.

PCR Amplification and Oligonucleotides-Single stranded cDNA was synthesized from 5 pg of poly(A+) RNA from TPA-treated LuSVX or 3T3 cells by reverse transcription using oligo(dT) as a primer (43) in a final volume of 50 pl. After reaction, the mixture was diluted 10-fold and 5 pl was used for the polymerase chain reaction using the following primers: coll-7, 5'-ATGAGAAAACCAAGATGTGGAGTGGAGTGCCT- GATGT-3' (corresponding to position 258 to 289 of rat collagenase; Ref. 44); coll-8, 5'-'ITGGCGGGGACGCCCA'ITITGATGATGATGA-3' (positions 570 to 601 of rat collagenase); coll-12, B'-AGACAGCATCTACTl" TGTCGCCAA'ITCCAGG-3' (positions 1259 to 1290 of rat collagenase);

CATGGGCAGCAAC-3' (positions 1259 to 1290 of rat collagenase); coll- 15, 5'-GGATATCTAGATGCGAAAGCCACGGTGCGGC(AiG)(T/A)CCC- AGA-3' (corresponding to positions 202 to 230 of human 72-kDa collagenase type IV, Ref. 45) and coll-16, 5"CCAGATCTCGAGTGGC- CGAACTCATGGGC(C/T)GC-CACG(C/A)G-3' (positions 1108 to 1137 of human 72-kDa collagenase type IV). Amplification was performed by 35 cycles (1 min at 94 "C, 2 min 45 "C, 3 min 72 "C) in a COY Temp-cycler. The resulting fragments were purified by gel electrophoresis on nonde- naturing 6% polyacrylamide gels followed by ligation in a pSP65-derived vector (pAZ vector). The cloned fragments were sequenced by standard procedures and analyzed by sequence comparison in the Gen- Bank nucleic acid sequence database. The newly identified murine stromelysin-2 cDNA and amino acid sequences were entered into the EMBL sequence Data Library (accession number X76537).

Induction of Exogenous Fos Expression in nansgenic Mice"F2 mice of line 41-4 (C57BL6 x SJL), bearing a 5.2-kb EcoRI Mx-c-fosD fragment (carrying a deletion of the 3'-untranslated region of c-fos to sta- bilize fos mRNA, Ref. 46) was injected with 800 pg of poly(1:C) (Phar- macia) to induce exogenous e-fos expression via induction of endogenous interferon production (47). Six h after injection of poly(1:C). organs were isolated and RNA was prepared as described (38). The transgenic mouse lines 506-1 and 505-11 constitutively express a transforming version of the c-fos gene under the control of the MHC class I promoter H2 (40).3

RESULTS

~011-13, 5"TCC'ITGGAGTGGTCCAGACGCAGGGAGTGGCCAAGCT-

Cloning of Mouse MMP-specific cDNA Sequences-To isolate appropriate probes for the analysis of MMP expression in mice we used NIH 3T3 cells and SV40 transformed mouse lung fibroblasts (LuSVX) treated with TPA for 10 h which express multiple forms of collagenolytic activity as measured by zymo- gram assay (data not shown). Poly(A+) RNA from these cells was used as a template for the "reverse transcriptase- polymerase chain reaction" procedure. The primers for the amplification of the reverse transcriptase products corresponded to regions of high homology (Fig. l a ) between human and rat collagenase I (coll 7/13; coll 8/12). The primers hybridized also to the corresponding rat stromelysin-1 and stromelysin-2 sequences. In addition, we used primers derived from the corresponding regions of the human 72-kDa collagenase IV sequence (coll 15/16). After PCR amplification we obtained multiple amplification products with templates from both 3T3 and LuSVX cells (data not shown). These products were subcloned in an pSP65 derived vector ( p M ) a n d sequenced. The regions of the MMP proteins encoded by the cloned cDNA fragments are sche- matically illustrated in Fig. la. The mouse collagenase I clone (730 base pairs) was found to contain a single nucleotide ex- change when compared to the mouse collagenase I cDNA isolated from mouse calvaria (Ref. 48; (G/A position 1239 in Ref. 51). The 935-base pair collagenase type IV fragment isolated from LuSVX cells was found to be totally identical with the recently isolated 72-kDa collagenase type IV cDNA from a mouse NIH 3T3 cell line (37) which exhibit 9 5 8 homology to the human gene in the region cloned. The 430-base pair stromelysin-1 cDNA sequence codes for an amino acid sequence identical with stromelysin-1 from mouse squamous cell carcinomas (49). An additional fragment of 730 base pairs encoding murine stromelysin-2 was isolated using cDNA from 3T3 cells. These newly identified cDNA and amino acid sequences were entered into the EMBL sequence Data Library (accession number

' U. Riither, unpublished data.

Fos-dependent Expression of Matrix Metalloproteinases in Adult Mice 10365

the murine MMP cDNAs isolated by PCR. FIG. 1. a , schematic representation of

The domain structure of MMPs (3) is shown on the top. Boxes below represent the regions covered by the isolated cDNAs. The catalytic domain containing the region required for Zn” binding is marked by an hatched box. 72-kDa collagenase N contains an additional domain that has similarities to the collagen-binding domain of fibronectin. Arrows indicate the locations of the oligonucleotides used for PCR. h, expression of mouse MMPs in tissues from adult mice. Two pg of highly purified poly(A’) RNA from different mouse tissues indicated on the top were used for a multiple tissue Northern (MTN, Clontech), and were sequentially hybridized with the cDNA probes encoding the various MMPs indicated on the right. According to the RNA size stan- dards marked on the filter the sizes of the specific transcripts are: 1.9 kb (stromelysin-1); 1.8 kb (stromelysin-2); 3.0 kb (72- kDa collagenase N); 2.8 kb (collagenase I). The different splice variants of p-actin are 1.6-2.0 kb in length. The presence of equivalent amounts of RNA from liver was confirmed by hybridization with a glyceraldehyde-3-phosphate dehydrogenase-specific probe (data not shown, see also MTN Clontech guidelines). Except for p-actin (1 day), x-ray films were exposed for 13-14 days. The specific activity of the different probes did not deviate by more than 2-fold.

a

X76537). The highest degree of homology (91%) was observed with stromelysin-2 from rat (11). Amino acid sequence comparison with mouse stromelysin-1 (49) and human stromelysin-3 (50) revealed 66 and 38% identity, respectively. Compared to mouse collagenase I, 65% homology was found (data not shown).

Tissue-specific Expression of MMPs-Due to differences in substrate specificity, the various MMPs are thought to have specific functions both in the physiological and pathological degradation of the ECM. Since different tissues greatly differ in their composition of the ECM, expression of the degrading enzymes may be regulated accordingly. So far, however, only the 72-kDa collagenase IV gene has been examined for its in vivo spatial expression (37). To get further support for differences in the in vivo function of the various MMPs, possibly reflected by differences in tissue-specific expression, the isolated cDNAs were used for Northern blot analysis (Fig.lb) using poly(A+) RNA from different tissues of adult mice (“multiple tissue Northern,” obtained from Clontech, see “Materials and Methods”).

Using the collagenase type IV-specific probe, a 3-kb transcript is detected in heart and lung. A weak signal is detected in skeletal muscle. In brain, spleen, liver, kidney, and testis, collagenase type IV seems not to be expressed at detectable levels. Interestingly, stromelysin-1 expression (1.9-kb transcript) exhibit the same type of tissue distribution. High levels of stromelysin-2 transcripts (1.8 kb) are found in heart and kidney. Ex- cept for testis, all other tissues examined show significant basal

STROMELYSIN 2 I J

COLLAGENASE I L I [I STROMELYSIN 1

i’\ COLLAGENASE IV

FIBRONECTIN

4- stromelysin

I i I c stromelysin

i

4- collagenase

1 E

4- collagenase

4- O - a c t i n

level expression. Low levels of collagenase I transcripts (2.8 kb) were observed in heart, spleen, and lung. No signal can be detected in brain, liver, and testis. Collagenase type I mRNA (2.8 kb) is abundantly expressed in muscle and kidney (Fig. l b ). In resolutions of total RNA collagenase type I transcripts are weakly detectable in kidney (Fig. 4) and bone (Figs. 3 and 4) indicating that muscle, kidney, and bone may be the most prominent sites of collagenase I expression in adult mice.

In summary, these results demonstrate that all four MMPs examined are expressed in a tissue-specific manner suggesting highly specific functions. Expression seems not to be coordinately regulated, perhaps with the exception of stromelysin-1 and collagenase IV in heart and lung. These results also show that, despite significant sequence homology, the various MMP cDNA probes do not cross-hybridize under these conditions.

Regulation of MMP Expression by Phorbol Esters, W Irra- diation, and M P - T o study the regulatory mechanisms of MMP transcription, we first analyzed the expression of collagenase I and IV, and stromelysin-1 and -2 in mouse fibroblasts in response to extracellular signals. Since the majority of extracellular agents elicit signal transduction pathways that in- volve either CAMP-dependent protein kinase A or protein kinase C, we analyzed MMP expression in mouse fibroblasts in response to CAMP or to the phorbol ester TPA known to activate protein kinase C (51). In addition, we used W irradiation as an example of a “stress response”-inducing agent that has been shown to induce collagenase I expression in human cells (10). RNA from rat fibroblasts transformed with activated ras served


r 1 stromelysin I

stromelysin 2

+ collagenase IV

stromelysin 2 !

I I

WV? GAPDH

FIG. 2. Regulation of mouse MMPs by TPA, C A M P , and UV irradiation. Ten pg of poly(A+) RNA from the various cells indicated on the top were used for Northern blot analysis. Cells were either left untreated (co), treated with CAMP or TPAfor 8 h (a) or irradiated with UV for the various time points indicated on the top in b. The mouse MMP signals are identical in size when compared to their rat homologous in ras-transformed rat2 fibroblasts. In the rat2 cells, an additional transcript of approximately 2.8 kb cross-hybridizes with the mouse stromelysin-1 and stromelysin-2 probes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for loading of equal amounts of RNA.

as positive control. Ras has been shown previously to induce enhanced levels of MMP transcripts (1114 RNA from undiffer- entiated F9 cells, which do not synthesize significant amounts of collagenolytic activity (data not shown and Ref. 36), were used as a negative control.

As shown in Fig. 2 a , expression of collagenase type I is very low in untreated mouse LuSVX fibroblasts. Upon treatment with TPA, expression is enhanced. In contrast, expression is not affected by CAMP. Expression is also enhanced by UV (Fig. 2b). However, like in human cells (lo), induction follows much slower kinetics than upon TPA treatment (lag period of 24 to 36 h). Induction by TPA and UV was also found for the stromely-

M. Hofmann, unpublished data.

sin-2 gene (Fig. 2). In the case of stromelysin-1, significant basal level expression was observed which was increased 2-3- fold after TPA treatment. Similar to collagenase type I, neither stromelysin-1 nor stromelysin-2 expression is increased in response to CAMP (Fig. 2a). Expression of the 72-kDa form of collagenase type IV was neither induced by TPA nor by CAMP (Fig. 2a) or UV (data not shown). In agreement with the lack of collagenolytic activity in F9 cells, we did not find detectable levels of transcripts encoding either one of these MMPs (Fig. 2a ).

Induction of Collagenase I Expression by c-Fos in Tkansgenic Mice-Based on studies of the human collagenase type I and stromelysin-1 promoters, the transcription factor AP-1, composed of members of the Fos and Jun proteins, was found to be the decisive factor in the regulation of collagenase expression in response to TPA and W in most cell culture systems (for review, see Ref. 23). Since expression of Fos was shown to be absolutely required for collagenase type I (31,32) and stromelysin-1 (301, and because induction of mouse collagenase (and stromelysin-2) by TPA and W (Fig. 2) closely resembles the expression found in human cells ( lo), we hypothesized that expression of Fos would also affect transcription of mouse MMPs. To confirm this assumption we analyzed MMP expression in transgenic mice in which c-fos is expressed under the control of the interferon-inducible M x promoter (47). In contrast to any type of cell culture system, this approach allows the analysis of Fos-dependent genetic and phenotypic responses in a more complex, intact multicellular organism. Upon treatment of the mice with poly(1:C) for 6 h, which induce the production of endogenous interferon, the expression of the fos transgene is strongly enhanced in almost all the tissues examined (Fig. 3) (47). Due to limitations in the amount of RNAfrom the various tissues, in non-induced mice, expression of collagenase type I was only detected in bone, but did not allow detection in spleen and kidney (Fig. 31, as found by using poly(A+) RNA (Fig. l b ). In the presence of enhanced levels of c-Fos, expression of collagenase type I becomes weakly detectable in spleen and liver; the most dominant enhancement is seen in bone (Fig. 3, right panel). Enhanced levels were also found in the thymus (data not shown). Despite vast overexpression of Fos in other tissues, collagenase I induction is restricted to bone, liver, spleen, and thymus (Fig. 3 and data not shown). In contrast, poly(1:C) treatment of mice which do not carry the transgene does not lead to enhanced collagenase type I expression (data not shown). The effect of Fos is specific for collagenase type I since it does neither induce collagenase type IV nor stromelysin-1 and stromelysin-2 (data not shown).

Expression of Collagenase I Is Constitutively Enhanced in Fos-induced Bone Tumors-The rapid tissue-specific increase in collagenase I expression in response to enhanced levels of c-Fos correlates with the sites of phenotypic alterations in transgenic mice which constitutively overexpress the transforming version of c-Fos (40). To determine whether Fos- induced collagenase I expression is only transient or whether induction is maintained to parallel Fos-induced cellular alterations we compared the amount of collagenase I transcripts in wild-type mice and in c-fos transgenic mice (506-1, 505-11; see also Ref. 40) at the stage of bone tumor formation.

As shown in Fig. 4 collagenase I expression is weakly detectable in kidney and bone but not in the brain of wild-type mice. In agreement with the lack of collagenase I induction following c-Fos expression in induced Mx-c-fos mice (Fig. 3), in the transgenic line 506-1 basal level expression of collagenase is not enhanced in kidney. No transcripts are detectable in the brain. In contrast, the amount of collagenase I transcripts is enhanced more than 200-fold in bone tumors of 506-1 mice. Identical results were observed in a second independent c-fos transgenic


1 Collagenase I

1 r- I I' c- fos

1

non-induced induced

FIG. 3. Enhanced expression of collagenase I in transgenic mice overexpressing c-Fos. Transgenic mice bearing an interferon-inducible Mx-c-fos expression vector were treated intraperitonally with poly(1:C) to induce c-fos expression via induction of endogenous interferon synthesis as described under "Materials and Methods." Six h after treatment, mice were killed and total RNA was prepared from different tissues indicated on the top. Expression of c-fos, collagenase I, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed by Northern blot analysis. Note that the relatively low expression of collagenase I in spleen (compared to the signal seen in Fig. l b ) is due to lower amounts of RNA in this sample (compare GAF'DH signal in non-induce2 and induced animalsj.

transgenic wildtype H2-cfos

r I" 1 I""

collagenase I (30 h)

collagenase I (7 d)

c - fos

GAPDH

FIG. 4 . Enhanced expression of collagenase I in Fos-induced bone tumors. Total RNA from kidney, brain, and bone was prepared from wild-type mice or from transgenic mice which constitutively overexpress c-fos (506-1), at the stage of bone tumor formation, and analyzed for expression of collagenase I, c-fos, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described in the legend to Fig. 3. To detect collagenase I in bone of 506-1 transgenic mice, the x-ray film was exposed for 30 h (top panel). To detect collagenase I in kidney and bone (in wild-type mice) the x-ray film was exposed for 7 days.

line 505-11 (data not shown). Enhanced expression of the c-fos transgene in these transgenic mice was detectable in all tissues examined (Fig. 4 and data not shown; see also Ref. 40).

These results demonstrate that induction of collagenase I by Fos does not follow a transient kinetic but seems to be maintained over a long period of time in a tissue-specific manner to parallel Fos-induced cellular alterations.

DISCUSSION

MMPs are a family of structurally related proteins involved in the degradation of the ECM which differ in regulation and substrate specificity (for review, see Refs. 1-5). These differ-

ences suggest very specific roles of individual MMPs both in the physiological and pathological degradation of the ECM. This assumption is strongly supported by our present study identi- fying differences in tissue-specific expression as well as differ- ential inducible transcription of MMP genes both in cultured mouse fibroblasts and in mice.

Tissue-specific Expression of Mouse MMPs-Using specific cDNAs encoding mouse collagenase I, 72-kDa collagenase IV, stromelysin-1 and stromelysin-2, it became clear that expression is not coordinately regulated. Exceptions to the rule are stromelysin-1 and 72-kDa collagenase IV, both of which are highly expressed in heart and lung, whereas in other tissues examined, only a very weak signal or no signal is detectable. Our data on collagenase IV are in good agreement with recent findings on the expression of this gene in newborn and adult mice (37). Tissue specificity of collagenase IV in heart and lung was explained by the accumulation of basement membranes in these tissues containing collagen type IV, the major substrate of collagenase type IV (45). Interestingly, stromelysin-1 was found to exhibit fairly high collagen IV degrading activity (approximately 50% of 72-kDa collagenase IV); much higher than stromelysin-2 (52), suggesting that both stromelysin-1 and collagenase IV may participate in collagen IV degradation. In agreement with the inability of both proteases to digest collagen type I, 11, and 111, the main substrates of collagenase type I (for review, see Refs. 1 3 ) , expression of collagenase type I is completely different from stromelysin-1 and collagenase type IV. Collagenase I is expressed predominantly in muscle and kidney. High basal level expression is also found in bone. This is not too surprising since collagenase type I activity has been suggested to play a decisive role in the remodeling of bone and cartilage (53, 541, which are composed primarily of collagen types I and 11, respectively (55). Expression of the various MMPs in different tissues, as shown in Fig. 1, however, does not necessarily reflect MMP activity. I t is possible, that tissue- specific differences in the expression of MMP inhibitors (TIMP-1, TIMP-2, TIMP-3; for review, see Refs. 1-5) or activa- tors of the proenzyme forms exist. In vitro studies using tissue culture systems revealed that stromelysin-1 in combination with plasmin or another proteinase is able to activate procollagenase I (17, 56, 57). However, in muscle and kidney, where very high levels of collagenase I transcripts are found, stromelysin-1 expression is not detectable, suggesting that a t


least in these tissues, stromelysin-1 is not involved in activation of procollagenase I but may be involved in the activation of latent 72-kDa collagenase IV, whose expression parallels stromelysin-1. Activation of latent collagenase I may be performed by stromelysin-2, which is expressed at significant levels in these tissues (Fig. 1). However, in vitro activation of procollagenase I by stromelysin-2 has yet to be demonstrated.

Dunscriptional Regulation of Mouse MMPs-The results on tissue-specific expression strongly suggest that transcription of the various MMP genes is regulated differently in response to extracellular signals. Our results on MMP expression in the mouse system are in agreement with previous studies on MMP expression in human, rat, and rabbit (9-11, 13, 15, 45, 58), showing that activation of protein kinase C (by treatment of cells with TPA) results in enhanced transcription of collagenase I, stromelysin-1, and stromelysin-2, but not 72-kDa collagenase Iv. In contrast, identical to rat and man, enhanced levels of CAMP neither induce stromelysin-1 and stromelysin-2 nor collagenase type I and type IV expression in mouse fibroblasts (9, 11,59). We also found strong enhancement of collagenase I and stromelysin-2 in response to UV irradiation. In the human cells, induction of collagenase I is considered as a hallmark of the induction of the mammalian stress response after treatment of cells with carcinogens (60). It is possible that, similar to human cells, induction is mediated by an UV-induced autocrine loop involving growth factors such as 11-la and basic fibroblast growth factor (34, 421, which explains the much slower time course of induction reaching a maximum between 36 and 48 h.

The strong conservation of MMP gene regulation across different species suggests that the cis-acting elements as well as the trans-acting factors regulating promoter activity of the MMP genes are also conserved. The most extensive studies on transcriptional regulation have been performed on the human collagenase I promoter. The transcription factor AP-1, composed of the products of members of the jun and fos gene families, has been shown to mediate enhanced transcription of collagenase in response to growth factors, cytokines, tumor promoters, carcinogens, and overexpression of certain onco- genes (for review, see Ref. 23). Transient transfection experi- ments including c-fos- or c-jun-specific RNA antisense sequences or transdominant-negative mutants of Fos and Jun confirmed the requirement of Fos and Jun for collagenase promoter activity (31, 32h5 Most recently, genomic sequences of the murine collagenase I gene were isolated and a high affinity AP-1 binding site that is preferentially recognized by Jun/Fos heterodimers was identified.6 Interestingly, the lack of mouse collagenase I expression in brain, liver, and testis (Fig. 1) correlates with the absence of c-jun expression in these tissues in mouse (61) or rat (62). On the other hand, high levels of c-jun transcripts are found in lung and heart (611, both of which exhibit basal level expression of collagenase I (Fig. 1). It will be interesting to examine c-jun expression in muscle, kidney, and bone where collagenase I is strongly expressed. However, it is likely that additional transcription factors binding to the collagenase I promoter, including members of the ets protein family (63), whose specific DNA recognition sequence is also present in the murine collagenase I promoter: may participate in promoter activity control.

MMP Expression in Fos Dansgenic Mice: A Causal Role of Collagenase Z in Establishing the Pathological Fos Pheno- type?-A more direct approach to demonstrate the role of AP-1 (JunlFos) in MMP expression was undertaken using a transgenic mouse system that allows efficient expression of c-Fos in almost any tissue. Since long-term overexpression of c-Fos re-

P. Angel, unpublished results. M. Schorpp, J. Schaper, I. Herr, and P. Angel, unpublished data.

sults in bone tumor formation (see below) we have first chosen an inducible expression vector system to monitor Fos-dependent immediate responses on MMP expression rather than possible secondary events caused by cell transformation. Short- term induction of c-Fos results in an increase of collagenase I mRNA in thymus, spleen, liver and, most dominantly, in bone. These data suggest that, similar to the human gene, the AP-1 site in the mouse collagenase I promoter is recognized by c-Fos (dimerized to Jun proteins) to mediate promoter activation. In agreement with the absence of an AP-1 binding site in the promoter of 72-kDa collagenase IV (at least in human; Ref. 64), c-Fos expression does not affect transcription of this gene. In- terestingly, stromelysin-1 and stromelysin-2, which both contain an AP-1 binding site in the promoter sequences (at least the rat genes; Refs. 9 and l l ) , are also not induced by enhanced levels of c-Fos. This is surprising since both genes are induced by TPA (Fig. 2). I t is possible that the mouse genes do not contain an AP-1 site and that TPA inducibility is mediated by other transcription factors. Alternatively, even in the presence of an AP-1 binding site, other transcription factors binding to the stromelysin-1 and stromelysin-2 promoters (which are possibly activated by TPA) may be required for enhanced transcription. This assumption is supported, first, by the findings that induction of rat stromelysin-1 by TPA requires the presence of additional growth factors (9); second, by the identifica- tion of c-Fos-dependent and e-Fos-independent pathways to regulate the stromelysin-1 promoter (30) and, third, by the fact that the AP-1 site in the human stromelysin-1 promoter, in contrast to collagenase I, is required for basal expression but is not necessary for the TPA response (65).

Most interestingly, Fos-dependent collagenase I expression is detectable only in bone, liver, spleen, and thymus. Other tissues, which greatly overexpress c-Fos, do not contain enhanced levels of collagenase I. The reason for the non-responsiveness in those tissues is presently unknown but may be explained by: (i) relatively low levels of Jun proteins, required for forming Fos/ Jun heterodimers capable of binding to the collagenase I promoter; (ii) high basal level expression of negatively acting members of the Fos and Jun protein families; e.g. JunB or JunD which are expressed at high levels in many tissues (611, or by the lack of appropriate post-translational modification of Jun and/or Fos (for review, see Ref. 23).

The tissue-specific restriction of collagenase I induction is particularly interesting with respect to the phenotypic conse- quences of long-term overexpression of c-Fos in transgenic mice, since collagenase I expression correlates with the sites of Fos-induced cellular alterations. Depending on the expression vector system used, disturbances of bone remodeling, formation of bone tumors, alterations in T cell development in the thymus, and enlarged spleens have been observed (38-40). In fact, we detect strongly enhanced collagenase I transcripts in the developing bone tumors, suggesting that, as a consequence of constitutive Fos expression, enhanced levels of collagenase I expression are maintained rather than following a transient kinetic of induction. Interestingly, such tumors were found to originate from osteoblasts (66) which are thought to be the sites of collagenase expression during bone remodeling (67,681. De- spite vast overexpression in other tissues, e.g. kidney, neither enhanced collagenase I mRNA levels nor tissue abnormalities have been detected by histological analysis. Despite the strik- ing correlation between collagenase expression and the sites of cellular alterations, a causal role of this enzyme during these processes has yet to be demonstrated, for example, in transgenic mice overexpressing collagenase in the bone (and spleen, liver, and thymus), or in mice in which the endogenous collagenase I loci have been inactivated by homologous recombina- tion. Of course many other genes regulated directly or indi-


rectly by Fos may be required for establishing and maintaining the phenotypical alterations described above. However, it is reasonable to assume that such genes are regulated in a Fos- dependent tissue-specific manner, similar to collagenase I.

Recently, Fos expression has been eliminated in mice and a retardation of animal growth and alterations in hematopoiesis as well as the development of osteopetrosis with deficiencies in bone remodeling and tooth eruption have been identified (69, 70). Using the various cDNAs described here encoding different MMPs, it may be possible to determine the causal role of Fos in tissue remodeling on a single cell level, e.g. by in situ hybridization. For example, whether the osteopetrotic phenotype in Fos-minus mice (69, 70) is characterized by the failure of osteo- clasts to resorb the bone and cartilage, or possibly by a lack of collagenase I production by osteoblasts (67, 68). On the other hand, transgenic mouse models expressing various transcription factors (including Fos and .hn), in which the members of the MMP protein family are differently expressed, may turn out to be powerful tools to identify the specific role of individual metalloproteinases during physiological and pathological processes.

Acknowledgments-We are grateful to H. Weiher for providing the LuSVX cell line, M. Hofmann for RNA from ras-transformed rat-2 fibroblasts, U. Giinthert for the pAZ plasmid, G. Murphy and M. Schorpp for helpful discussion, and P. Herrlich for critical reading of the manu- script.

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