- Email: [email protected]
Regulation of MMP-2 Gene Transcription in Dermal Wounds
ORIGINAL ARTICLE
Regulation of MMP-2 Gene Transcription in Dermal Wounds Petra Lynen Jansen1,2, Raphael Rosch2, Marc Jansen2, Marcel Binnebo¨sel2, Karsten Junge2, Alexandra Alfonso-Jaume3, Uwe Klinge2,4, David H. Lovett5 and Peter R. Mertens6 Matrix metalloproteinase-2 (MMP-2, gelatinase A) plays an essential role in angiogenesis, inflammation, and fibrosis. These processes are critical for wound healing and accordingly elevated levels of MMP-2 expression have been detected after skin injury. Our goal was to investigate the transcriptional activation of the MMP-2 gene in a model of skin injury by using two different MMP-2/LacZ-reporter mice. Upon skin injury MMP-2 expression was upregulated, whereas tissue from normal skin stained negative except for occasional macrophages, sweat glands, and hair follicles. Skin injury also activated MMP-2 proteolytic activity and reporter gene expression. We demonstrate that MMP-2 regulatory sequences 1686/ þ 423 drive appropriate injury-induced MMP-2-promoter activation. Reporter gene expression was predominantly detectable in endothelial cells and in macrophages. Deletion of the 50 responsive element, denoted RE-1, residing at 1241/ þ 423 bp of the regulatory sequence led to abrogated MMP-2 transcription in vivo. The findings define a crucial role for the enhancer element RE-1 in injury-induced MMP-2 transcription of the skin. Journal of Investigative Dermatology (2007) 127, 1762–1767; doi:10.1038/sj.jid.5700765; published online 8 March 2007
INTRODUCTION Tissue injury initiates an inflammatory response, tissue formation, and remodelling to reconstruct the wounded area. Genetically modified animal models have helped to define the roles of many key molecules in these processes (Werner and Grose, 2003; Grose and Werner, 2004). As such, matrix metalloproteinases (MMPs) have been identified as key enzymes that regulate wound healing and matrix remodelling (Kato et al., 2001a). These zinc-dependent enzymes operate by hydrolyzing components of the extracellular matrix and by directly affecting cellular phenotypes, proliferation rates, and the inflammatory reaction (Marti et al., 1994; Turck et al., 1996). Inhibition of MMPs by a broad MMP inhibitor (GM6001) prevents the MMP-related promotion of wound healing via a complex mechanism involving stimulation of angiogenesis and inhibition of inflammation (Jackson et al., 2005). In diseases such as arthritis (Ishikawa et al., 2005), cancer (Mandal et al., 2003), atheroma (Wu and Li, 2006), 1
Interdisciplinary Centre for Clinical Research BIOMAT, University Hospital, RWTH Aachen, Aachen, Germany; 2Department of Surgery, University Hospital, RWTH Aachen, Aachen, Germany; 3Department of Medicine, Williams Middleton Memorial, University of Wisconsin, Madison, Wisconsin, USA; 4Institute for Applied Medical Engineering, Helmholtz Institute, University Hospital, RWTH Aachen, Aachen, Germany; 5Department of Medicine, San Francisco Veterans Affairs Medical Centre, University of California, San Francisco, California, USA and 6Departments of Nephrology and Clinical Immunology, University Hospital Aachen, RWTH Aachen, Aachen, Germany Correspondence: Dr Petra Lynen Jansen, Interdisciplinary Centre for Clinical Research BIOMAT, University Hospital, RWTH Aachen, Pauwelsstr. 30, Aachen 52074, Germany. E-mail: [email protected] Received 7 August 2006; revised 15 December 2006; accepted 15 December 2006; published online 8 March 2007
1762 Journal of Investigative Dermatology (2007), Volume 127
and tissue ulceration (Agren, 1994), MMP-2 (72 kDa collagenase, gelatinase A) enzymatic activities are upregulated at the sites of inflammation, fibrosis, and angiogenesis. In MMP-2-deficient mice, impaired neoangiogenesis was observed in the presence of tumor cells (Itoh et al., 1998) or in experimental inflammation-associated corneal neovascularization (Kato et al., 2001a; Samolov et al., 2005). MMP2 expression is absent in healthy skin except for occasional macrophages, sweat glands, and hair follicles (Agren, 1994; Vaisanen et al., 1996). Mice with targeted deletion of the MMP-2 gene exhibit only slight disturbances of growth indicating that lack of MMP-2 is not critical under physiological conditions. However, these mice demonstrated diminished angiogenesis after injury (Kato et al., 2001b). The extent of MMP-2 expression and enzymatic activity is regulated at the transcriptional, translational, and posttranslational levels (Overall and Lopez-Otin, 2002). Regulatory elements spanning the proximal 1686 bp of the MMP-2 gene relative to the translational start have been shown to govern MMP-2 gene transcription (Frisch and Morisaki, 1990; Harendza et al., 1995; Mertens et al., 1997, 1998, 2002; Harendza et al., 2000). A strong enhancer element, denoted response element-1 (RE-1), is located at 1322/1282 bp of the rat gene (Harendza et al., 1995) that is evolutionarily conserved and is similarly operative within the human gene at 1657/1619 bp relative to the transcription start site (Mertens et al., 1999). At least five distinct transcription factors may, mostly cooperatively, bind to the RE-1. Transcriptional regulation of MMP-2 synthesis in vivo is similarly coordinated by distinct subsets of transcription factors, as shown by a recent study on ischemia of the limb (Lee et al., 2005). & 2007 The Society for Investigative Dermatology
P Lynen Jansen et al. Regulation of MMP-2 Gene Transcription in Dermal Wounds
Given these findings that support the pivotal role of MMP2 enzyme in wound healing and inflammatory response, we set out to unravel the molecular mechanisms that govern MMP-2 gene transcription in vivo. A wound-healing model was established in transgenic reporter mice harboring a b-galactosidase-reporter gene (LacZ), driven by defined regulatory sequences of the MMP-2 gene. Furthermore, the spatial and temporal transcriptional regulation of the MMP-2 gene was analyzed at the cellular level and compared with protein synthesis and enzymatic activity. RESULTS MMP-2 protein expression and MMP proteolytic activity is enhanced in healing wounds
To determine patterns of MMP-2 expression in healthy skin and after full-thickness dermal incision, immunohistochemistry was performed with MMP-2-specific antibodies. In healthy skin, MMP-2 protein expression was detected in the epidermal layer and in hair follicles (Figure 1a). Quantitative analysis of MMP-2-positive cells demonstrated that MMP-2 protein expression was significantly enhanced on days 7, 21, and 90 after injury compared with non-incised skin (Figure 1b and c). In addition to macrophages and fibroblasts, vessels within the scar tissue exhibited MMP-2 positivity (Figure 1d). To corroborate MMP-2 proteolytic activity with expression levels, results obtained by immunohistochemistry were compared with in situ zymograms. Although basal levels of MMP-2 protein expression were detected in healthy skin, proteolytic activity was nearly absent in in situ zymograms (Figure 2a and c). After injury, gelatinolytic activity could be visualized (translucent areas in Figure 2b). Semiquantitative
a
% MMP-2 positive cells/0.01 mm2
c
analysis of in situ zymography revealed enhanced MMP proteolytic activity at all time points in healing wounds. No gelatinolytic activity was detected when MMPs were blocked by a synthetic inhibitor (Figure 2d). In summary, MMP-2 protein expression and proteolytic activity are upregulated in healing wounds. MMP-2 transcription is induced via regulatory sequences 1686/ þ 423 in healing wounds
It is known that MMP-2 protein synthesis is regulated at the level of transcription as well as translation (Corcoran et al., 1996). Distinct regulatory elements within the MMP-2 gene promoter have been mapped previously, as outlined in the Introduction. In vitro, these regulatory elements confer cellspecific gene transcription. Transcriptional activity conferred by sequences 1686/ þ 423 was assessed in F8 animals (Figure 3a). Control-staining was performed by omitting the primary antibody (Figure 3b). Figure 3c illustrates dermal MMP-2-reporter gene expression in F8 mice 7 days after injury: Within the scar tissue, b-galactosidase-reporter gene expression was significantly enhanced. In healthy skin, b-galactosidase staining was restricted to the epidermal layer and hair follicles. The reporter-gene expression driven by MMP-2 regulatory sequences 1686/ þ 413 was upregulated over time in F8 mice (Figure 4a). Furthermore, b-galactosidase staining was obviously enhanced in F8 animals when compared with F8del animals with selectively deleted sequences 1686/ þ 1241 (Figure 4a and b). These differences in b-galactosidase expression patterns could be observed at all time points. The decreased reporter-gene expression in F8del mice indicates that RE-1 acts as an
b
50 m
60
d
50 40 30 20 10 0
Skin
Day 7
Day 21
Day 90 25 m
Figure 1. MMP-2 protein expression is enhanced in healing wounds. Immunohistochemistry for MMP-2 protein was performed and analyzed quantitatively for time points 7, 21, and 90 days. (a) MMP-2 protein expression in healthy skin is detectable within the epidermal layer, hair follicles, and in several cells interspersed in the subepidermal layer. Morphologically, these cells correspond to macrophages and fibroblasts. (b) MMP-2 protein expression is strongly enhanced 7 days after full dermal-skin incision. Bar ¼ 50 mm. (c) Morphometric analyses revealed significantly enhanced MMP-2 protein expression at all time points in healing wounds when compared with healthy skin. Tissue areas of 0.01 mm2 were analyzed in F8 and F8del transgenic mice (n ¼ 12 at each time point). Data are represented as means 7SEM. Differences between both strains were not detected (data not presented). (d) MMP-2 expression in vessel walls. Section of (b) is presented.
www.jidonline.org 1763
P Lynen Jansen et al. Regulation of MMP-2 Gene Transcription in Dermal Wounds
a
b
c
d Strong
Score
3
Moderate
2
Weak
1
Skin
0
Day 7
Day 21
Day 90
50 m
Figure 2. MMP proteolytic activity is enhanced in healing wounds. In situ zymography was performed and analyzed quantitatively for 7, 21, and 90 days. Translucent areas correspond to gelatinolytic activity. (a) Absence of proteolytic activity in healthy skin. (b) Areas of proteolytic activity 7 days after dermal incision, indicated by black arrows. (c) Semiquantitative analysis of in situ zymography reveals enhanced MMP proteolytic activity at all time points in healing wounds when compared with healthy skin. Tissue areas were analyzed in F8 and F8del transgenic mice and summarized (n ¼ 12 at each time point). Data are represented as means 7SEM. Differences between both strains were not detected (data not presented). (d) Control experiments with MMP-inhibitor 1. 10-phenanthroline were performed with specimen on day 7 after dermal incision. Bar ¼ 50 mm.
a
b
F8 + 423
−1686
Control staining
Lacz
Response element 1
c
Skin
Scar
Skin
200 m
Figure 3. MMP-2 transcription is induced via regulatory sequences 1686/ þ 423 in healing wounds. Immunohistochemistry for b-galactosidase was performed in F8 mice. (a) Schematic depiction of transgene introduced into F8 mice. LacZ-reporter gene is driven by MMP-2 regulatory sequences 1686/ þ 413 including the RE-1. (b) Control stainings were performed by omitting the primary anti-b-galactosidase antibody. (c) b-galactosidasereporter gene expression in F8 mice was significantly enhanced within the scar tissue. In contrast, b-galactosidase staining was restricted to the epidermal layer and hair follicles in healthy skin. A representative image taken with specimen from day 7 following dermal incision is shown. Bar ¼ 200 mm.
enhancer element of MMP-2 transcription in healing wounds in vivo. Localization of MMP-2 transcription
By means of b-galactosidase immunofluorescence, the distribution of MMP-2 transcription within the tissue specimens was further analyzed. As seen by immunohistochemistry, basal staining for b-galactosidase was detectable in hair follicles, within the epidermis and occasionally in fibroblasts 1764 Journal of Investigative Dermatology (2007), Volume 127
and residential macrophages in non-incised skin of F8 mice (Figure 5a). After injury, cells with intense b-galactosidase staining were evident (Figure 5b, white arrows). These cells were absent in F8del mice, whereas basal staining of b-galactosidase in hair follicles and within the epidermis still occurs (Figure 5c). Immunofluorescence costaining for b-galactosidase and CD68 or vWF revealed that MMP-2 regulatory sequence 1686/ þ 413, which includes the RE-1, is required for MMP-2 transcription in macrophages and vascular endothelial cells, respectively (Figure 6a and b). DISCUSSION In this report, we provide insights into the transcriptional regulation of MMP-2 expression during the course of dermalwound healing. We show that MMP-2 expression and proteolytic activity is upregulated in the subepidermal layer following skin injury. The results obtained by immunohistochemistry indicate an induction of MMP-2 protein expression over time, whereas MMP proteolytic activities were highest on day 7 and declined over time. It is likely that MMP proteolytic activity is subjected to regulatory interaction with tissue inhibitor of metalloproteinases or microtubule–MMPs. Ongoing MMP-2 protein synthesis provides a pool of latent enzyme ready for subsequent activation. To date, several therapeutic approaches have been taken to chemically block MMP activities at the protein level, but clinical trials with variably selective MMP inhibitors have been mostly unsuccessful. Our findings indicate that decreased transcriptional activity occurs following partial deletion of the RE-1. These data indicate that the RE-1 is an important cis-acting factor in injury related dermal-wound healing. In this regard, selective inactivation of MMP-2specific regulatory elements may be more promising. Such an
P Lynen Jansen et al. Regulation of MMP-2 Gene Transcription in Dermal Wounds
a
Day 7
Day 21
Day 90
F8 −1686 Response element 1
+ 423
Lacz
b F8del −1241
+ 423
Lacz 100 m
Figure 4. The MMP-2-regulatory sequences 1686/1241 are critically involved in conferring MMP-2 transcription in healing wounds. Immunohistochemical staining for b-galactosidase in F8 mice was compared with staining in F8del mice for time points 7, 21, and 90 days. (a) Reporter gene expression driven by MMP-2 regulatory sequences 1686/ þ 413 is induced over time in F8 mice. (b) The reporter gene expression is significantly reduced after deletion of regulatory sequences 1686/1241 at all time points in F8del mice. Bar ¼ 100 mm.
a
F8, skin (including RE-1)
-Galactosidase (red) DAPI (blue)
b
F8, scar (including RE-1)
c
F8del, scar
a
CD 68
-Galactosidase
DAPI
b
vWF
-Galactosidase
DAPI
(RE-1 deleted)
100 m
Figure 5. Localization of MMP-2 transcription is dependent on RE-1. Immunofluorescence staining for b-galactosidase (red) was performed. Nuclei were detected by DAPI staining (blue). (a) In healthy skin, staining for b-galactosidase was predominantly detected in hair follicles (F), within the epidermis (E) and occasionally in fibroblasts and residential macrophages within the dermis. (b) After dermal incision, areas of intense b-galactosidase expression within the dermis were detectable in F8 mice (white arrows). (c) Areas of intense b-galactosidase staining within the dermis were absent in F8del mice, whereas b-galactosidase staining was still detectable in hair follicles (F) and within the epidermis (E). Bar ¼ 100 mm.
approach was recently realized by Miyake et al. (2006), who successfully prevented the development of arterial aneurysm via blockage of MMP-2 transcription by NF-kB/ets1 oligonucleotide decoys in rabbits. The identification of RE-1 as a key regulatory element in MMP-2 transcription following dermal injury may provide a similar basis for the application of double-stranded oligodeoxynucleotides that are directed against one or even more specific cis-elements. Moreover, our data indicate that MMP-2 gene transcription was cell specifically regulated. We identified sequences 1686/1241 as critical for enhancement of MMP-2 transcription in macrophages and vascular endothelial cells in vivo. It is known that macrophages are strongly involved in MMP-2 expression and gene regulation at sites of inflammation. In vivo, Eerola et al. observed a direct relation between
50 m
Figure 6. MMP-2-regulatory sequences 1686/ þ 413, including RE-1, drive MMP-2 transcription in macophages and vascular endothelial cells. Immunofluorescence costaining of b-galactosidase and CD68 or vWF in F8 mice. (a) CD68 (red) positive macrophages exhibited b-galactosidase (green) costaining; (b) vWF (red) positive vascular endothelial cells exhibited b-galactosidase (green) costaining. Bar ¼ 50 mm.
MMP-2 gene activation and porcine posttransplant fibrotic reaction (Eerola et al., 2005). Modulation of MMP-2 expression might represent a novel approach for reorganizing cell–cell interactions with the aim to optimize wound healing or inflammation-associated diseases. Potential therapeutic targets are the transcription factors, for example Y-box protein-1 (YB-1), that bind to the RE-1 and cell-specifically trans-activates as well as transrepresses the MMP-2 promoter (Mertens et al., 1997). Interference with MMP-2 transcription may provide for cellspecific fine-tuning of the MMP-promoter transcriptional regulation. In conclusion, our findings highlight the complexity of MMP-2 gene regulation following dermal wounding, a key molecule for matrix remodelling. The established transgenic mice serve as useful models for further in vivo www.jidonline.org 1765
P Lynen Jansen et al. Regulation of MMP-2 Gene Transcription in Dermal Wounds
testing of ‘‘optimized’’ wound healing and neoangiogenesis with the aim to disrupt and/or enhance MMP-2 transcription. MATERIALS AND METHODS MMP-2/LacZ transgenic mice model The two mice strains used as animal models have been described in detail (Lee et al., 2005). In brief, mouse strain F8 harbor a aˆ-galactosidase-reporter gene (LacZ) under control of MMP-2 regulatory sequences 1686/ þ 423 that extend to the middle of the second exon. To determine the significance of the RE-1 that extends from 1282 to 1322 bp, F8del mice were utilized that harbor MMP2-regulatory sequence 1241/ þ 423, excluding the RE-1.
Study design and surgery The mice were fixed in the supine position. Full-thickness dermal incisions extending over 1.5 cm were performed 1 cm bilateral of the abdominal midline in 18 animals of each strain. The surgical procedure was performed under sterile conditions and general anesthesia by intramuscular administration of ketamine (ketamin 10%; Sanofi-Ceva, Du¨sseldorf, Germany) and xylazine (rompun 2%; Bayer, Leverkusen, Germany). After 7, 21, and 90 days of implantation, animals were killed by isoflurane asphyxation and decapitation. Tissue was harvested, split, and either fixed in 10% formalin for morphological analysis or quick-frozen and stored at 801C for activity assays. All animals received human care in accordance with the guidelines of the ‘‘Deutsche Tierschutzgesetz’’ (50.203.2-AC 46, 38/02) and the National Institutes of Health guidelines for the use of laboratory animals.
Histomorphological and immunohistochemical analyses Histological and immunohistochemical investigations were performed on paraffin-embedded 3-mm sections using peroxidaseconjugated, affinity-isolated Igs. All sections were routinely stained with hematoxylin and eosin and were processed at the same time to reduce internal staining variations or histomorphological studies. Immunohistochemistry was performed using the avidin–biotin complex and diaminobenzidine as chromogen. To detect cells with MMP-2-promoter activity, sections were incubated with monoclonal anti-b-galactosidase antibody (Europa, Cambridge, UK, 1:1700) followed by biotinylated rabbit anti-mouse IgG (Dako, Denmark, 1:300). Immunohistochemical staining was explored for MMP-2 (polyclonal rabbit, Biomol, Hamburg, 1:1000), a-smooth muscle actin (polyclonal rabbit, Abcam, Cambridge, UK, 1:300), CD68 (polyclonal rabbit, Santa Cruz, USA, 1:100), and vWF (polyclonal, Dako, Denmark, 1:400) followed by incubation with goat anti-rabbit IgG antibody (Dako, Denmark, 1:500). Coexpression of b-galactosidase with MMP-2, a-smooth muscle actin, CD68, and vWF was determined by immunofluorescence using goat anti-mouse, donkey anti-goat, and goat anti-rabbit secondary antibodies (Alexa Fluor 488, Molecular probes, Eugene, OR). Semiquantitative analyses of immunohistochemical staining (area 100 100 mm, 400-fold magnification) were performed with digital imaging analyzing software (Image-Pro Plus. Media Cybernetics, Silver Spring, MD). Control stainings were performed by omitting the primary antibodies.
Zymography Serial-frozen sections were placed on MMP in situ ZymoFilms composed of a 7-mm-thick layer of gelatin on a polyester base (Wako 1766 Journal of Investigative Dermatology (2007), Volume 127
Chemicals, Neuss, Germany) or MMP-PT in situ ZymoFilm, which contains the MMP inhibitor 1.10-phenanthroline. Films were incubated at 371C for 24 hours and stained with Biebrich Scarlet (Wako Chemical Industries, Japan). Gelatin digestion is visualized as transparency. Results obtained by comparison of both films indicate localization of MMP-specific proteolytic activity. Two blinded observers evaluated the gelatinolytic activity according to a scoring system (: negative; þ : weak; þ þ : moderate; þ þ þ : strong) in six independent tissue samples for each subgroup and time point.
Statistics The study was performed as balanced incomplete block design. Data were organized according to time, strain, and side. Differences between the groups were evaluated for statistical significance by analysis of variance describing the effect of strain, day, strain day, and side. Data were described as mean values 7SD. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft, DFG grant KL 1320/2-1 (UK, P.R.M.) and JA 1123/1-1 (P.L.J., UK, P.R.M., M.J.), SFB 542 project C4 (P.R.M.), IZKF-BIOMAT, RWTH-Aachen, Project no. NTV 41 and NIH grant DK039776 (D.H.L.).
REFERENCES Agren MS (1994) Gelatinase activity during wound healing. Br J Dermatol 131:634–40 Corcoran ML, Hewitt RE, Kleiner DE Jr, Stetler-Stevenson WG (1996) MMP-2: expression, activation and inhibition. Enzyme Protein 49:7–19 Eerola LM, Alho HS, Maasilta PK, Inkinen KA, Harjula AL, Litmanen SH et al. (2005) Matrix metalloproteinase induction in post-transplant obliterative bronchiolitis. J Heart Lung Transplant 24:426–32 Frisch SM, Morisaki JH (1990) Positive and negative transcriptional elements of the human type IV collagenase gene. Mol Cell Biol 10:6524–32 Grose R, Werner S (2004) Wound-healing studies in transgenic and knockout mice. Mol Biotechnol 28:147–66 Harendza S, Lovett DH, Stahl RAK (2000) The hematopoietic transcription factor PU.1 represses gelatinase A transcription in glomerular mesangial cells. J Biol Chem 275:19552–9 Harendza S, Pollock AS, Mertens PR, Lovett DH (1995) Tissue-specific enhancer-promoter interactions regulate high level constitutive expression of matrix metalloproteinase 2 by glomerular mesangial cells. J Biol Chem 270:18786–96 Ishikawa T, Nishigaki F, Miyata S, Hirayama Y, Minoura K, Imanishi J et al. (2005) Prevention of progressive joint destruction in collagen-induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR255031. Br J Pharmacol 144:133–43 Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, Itohara S (1998) Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res 58:1048–51 Jackson CJ, Xue M, Thompson P, Davey RA, Whitmont K, Smith S et al. (2005) Activated protein C prevents inflammation yet stimulates angiogenesis to promote cutaneous wound healing. Wound Repair Regen 13:284–94 Kato T, Kure T, Chang JH, Gabison EE, Itoh T, Itohara S et al. (2001a) Diminished corneal angiogenesis in gelatinase A-deficient mice. FEBS Lett 508:187–90 Kato T, Kure T, Chang JH, Gabison EE, Itoh T, Itohara S et al. (2001b) Diminished corneal angiogenesis in gelatinase A-deficient mice. FEBS Lett 508:187–90
P Lynen Jansen et al. Regulation of MMP-2 Gene Transcription in Dermal Wounds
Lee JG, Dahi S, Mahimkar R, Tulloch NL, Alfonso-Jaume MA, Lovett DH et al. (2005) Intronic regulation of matrix metalloproteinase-2 revealed by in vivo transcriptional analysis in ischemia. Proc Natl Acad Sci USA 102:16345–50 Mandal M, Mandal A, Das S, Chakraborti T, Chakraborti S (2003) Clinical implications of matrix metalloproteinases. Mol Cell Biochem 252:305–29 Marti HP, Lee L, Kashgarian M, Lovett DH (1994) Transforming growth factorbeta 1 stimulates glomerular mesangial cell synthesis of the 72-kd type IV collagenase. Am J Pathol 144:82–94 Mertens PR, Alfonso-Jaume MA, Steinmann K, Lovett DH (1998) A synergistic interaction of transcription factors AP2 and YB-1 regulates gelatinase A enhancer-dependent transcription. J Biol Chem 273:32957–65 Mertens PR, Alfonso-Jaume MA, Steinmann K, Lovett DH (1999) YB-1 regulation of the human and rat gelatinase A genes via similar enhancer elements. J Am Soc Nephrol 10:2480–7 Mertens PR, Harendza S, Pollock AS, Lovett DH (1997) Glomerular Mesangial Cell-specific Transactivation of Matrix Metalloproteinase 2a´Transcription Is Mediated by YB-1. J Biol Chem 272:22905–12 Mertens PR, Steinmann K, Alfonso-Jaume MA, En-Nia A, Sun Y, Lovett DH (2002) Combinatorial interactions of p53, AP2 and YB-1 with a single enhancer element regulate gelatinase A expression in neoplastic cells. J Biol Chem 277:24875–82
Miyake T, Aoki M, Nakashima H, Kawasaki T, Oishi M, Kataoka K et al. (2006) Prevention of abdominal aortic aneurysms by simultaneous inhibition of NF[kappa]B and ets using chimeric decoy oligonucleotides in a rabbit model. Gene Ther 13:695–704 Overall CM, Lopez-Otin C (2002) Strategies for MMP inhibition in cancer: innovations for the post-trial era. Nat Rev Cancer 2:657–72 Samolov B, Steen B, Seregard S, van dP I, Montan P, Kvanta A (2005) Delayed inflammation-associated corneal neovascularization in MMP-2-deficient mice. Exp Eye Res 80:159–66 Turck J, Pollock AS, Lee LK, Marti HP, Lovett DH (1996) Matrix metalloproteinase 2 (gelatinase A) regulates glomerular mesangial cell proliferation and differentiation. J Biol Chem 271:15074–83 Vaisanen A, Tuominen H, Kallioinen M, Turpeenniemi-Hujanen T (1996) Matrix metalloproteinase-2 (72 kD type IV collagenase) expression occurs in the early stage of human melanocytic tumour progression and may have prognostic value. J Pathol 180:283–9 Werner S, Grose R (2003) Regulation of wound healing by growth factors and cytokines. Physiol Rev 83:835–70 Wu M, Li YG (2006) The expression of CD40-CD40L and activities of matrix metalloproteinases in atherosclerotic rats. Mol Cell Biochem 282:141–6
www.jidonline.org 1767
Regulation of MMP-2 Gene Transcription in Dermal Wounds Petra Lynen Jansen1,2, Raphael Rosch2, Marc Jansen2, Marcel Binnebo¨sel2, Karsten Junge2, Alexandra Alfonso-Jaume3, Uwe Klinge2,4, David H. Lovett5 and Peter R. Mertens6 Matrix metalloproteinase-2 (MMP-2, gelatinase A) plays an essential role in angiogenesis, inflammation, and fibrosis. These processes are critical for wound healing and accordingly elevated levels of MMP-2 expression have been detected after skin injury. Our goal was to investigate the transcriptional activation of the MMP-2 gene in a model of skin injury by using two different MMP-2/LacZ-reporter mice. Upon skin injury MMP-2 expression was upregulated, whereas tissue from normal skin stained negative except for occasional macrophages, sweat glands, and hair follicles. Skin injury also activated MMP-2 proteolytic activity and reporter gene expression. We demonstrate that MMP-2 regulatory sequences 1686/ þ 423 drive appropriate injury-induced MMP-2-promoter activation. Reporter gene expression was predominantly detectable in endothelial cells and in macrophages. Deletion of the 50 responsive element, denoted RE-1, residing at 1241/ þ 423 bp of the regulatory sequence led to abrogated MMP-2 transcription in vivo. The findings define a crucial role for the enhancer element RE-1 in injury-induced MMP-2 transcription of the skin. Journal of Investigative Dermatology (2007) 127, 1762–1767; doi:10.1038/sj.jid.5700765; published online 8 March 2007
INTRODUCTION Tissue injury initiates an inflammatory response, tissue formation, and remodelling to reconstruct the wounded area. Genetically modified animal models have helped to define the roles of many key molecules in these processes (Werner and Grose, 2003; Grose and Werner, 2004). As such, matrix metalloproteinases (MMPs) have been identified as key enzymes that regulate wound healing and matrix remodelling (Kato et al., 2001a). These zinc-dependent enzymes operate by hydrolyzing components of the extracellular matrix and by directly affecting cellular phenotypes, proliferation rates, and the inflammatory reaction (Marti et al., 1994; Turck et al., 1996). Inhibition of MMPs by a broad MMP inhibitor (GM6001) prevents the MMP-related promotion of wound healing via a complex mechanism involving stimulation of angiogenesis and inhibition of inflammation (Jackson et al., 2005). In diseases such as arthritis (Ishikawa et al., 2005), cancer (Mandal et al., 2003), atheroma (Wu and Li, 2006), 1
Interdisciplinary Centre for Clinical Research BIOMAT, University Hospital, RWTH Aachen, Aachen, Germany; 2Department of Surgery, University Hospital, RWTH Aachen, Aachen, Germany; 3Department of Medicine, Williams Middleton Memorial, University of Wisconsin, Madison, Wisconsin, USA; 4Institute for Applied Medical Engineering, Helmholtz Institute, University Hospital, RWTH Aachen, Aachen, Germany; 5Department of Medicine, San Francisco Veterans Affairs Medical Centre, University of California, San Francisco, California, USA and 6Departments of Nephrology and Clinical Immunology, University Hospital Aachen, RWTH Aachen, Aachen, Germany Correspondence: Dr Petra Lynen Jansen, Interdisciplinary Centre for Clinical Research BIOMAT, University Hospital, RWTH Aachen, Pauwelsstr. 30, Aachen 52074, Germany. E-mail: [email protected] Received 7 August 2006; revised 15 December 2006; accepted 15 December 2006; published online 8 March 2007
1762 Journal of Investigative Dermatology (2007), Volume 127
and tissue ulceration (Agren, 1994), MMP-2 (72 kDa collagenase, gelatinase A) enzymatic activities are upregulated at the sites of inflammation, fibrosis, and angiogenesis. In MMP-2-deficient mice, impaired neoangiogenesis was observed in the presence of tumor cells (Itoh et al., 1998) or in experimental inflammation-associated corneal neovascularization (Kato et al., 2001a; Samolov et al., 2005). MMP2 expression is absent in healthy skin except for occasional macrophages, sweat glands, and hair follicles (Agren, 1994; Vaisanen et al., 1996). Mice with targeted deletion of the MMP-2 gene exhibit only slight disturbances of growth indicating that lack of MMP-2 is not critical under physiological conditions. However, these mice demonstrated diminished angiogenesis after injury (Kato et al., 2001b). The extent of MMP-2 expression and enzymatic activity is regulated at the transcriptional, translational, and posttranslational levels (Overall and Lopez-Otin, 2002). Regulatory elements spanning the proximal 1686 bp of the MMP-2 gene relative to the translational start have been shown to govern MMP-2 gene transcription (Frisch and Morisaki, 1990; Harendza et al., 1995; Mertens et al., 1997, 1998, 2002; Harendza et al., 2000). A strong enhancer element, denoted response element-1 (RE-1), is located at 1322/1282 bp of the rat gene (Harendza et al., 1995) that is evolutionarily conserved and is similarly operative within the human gene at 1657/1619 bp relative to the transcription start site (Mertens et al., 1999). At least five distinct transcription factors may, mostly cooperatively, bind to the RE-1. Transcriptional regulation of MMP-2 synthesis in vivo is similarly coordinated by distinct subsets of transcription factors, as shown by a recent study on ischemia of the limb (Lee et al., 2005). & 2007 The Society for Investigative Dermatology
P Lynen Jansen et al. Regulation of MMP-2 Gene Transcription in Dermal Wounds
Given these findings that support the pivotal role of MMP2 enzyme in wound healing and inflammatory response, we set out to unravel the molecular mechanisms that govern MMP-2 gene transcription in vivo. A wound-healing model was established in transgenic reporter mice harboring a b-galactosidase-reporter gene (LacZ), driven by defined regulatory sequences of the MMP-2 gene. Furthermore, the spatial and temporal transcriptional regulation of the MMP-2 gene was analyzed at the cellular level and compared with protein synthesis and enzymatic activity. RESULTS MMP-2 protein expression and MMP proteolytic activity is enhanced in healing wounds
To determine patterns of MMP-2 expression in healthy skin and after full-thickness dermal incision, immunohistochemistry was performed with MMP-2-specific antibodies. In healthy skin, MMP-2 protein expression was detected in the epidermal layer and in hair follicles (Figure 1a). Quantitative analysis of MMP-2-positive cells demonstrated that MMP-2 protein expression was significantly enhanced on days 7, 21, and 90 after injury compared with non-incised skin (Figure 1b and c). In addition to macrophages and fibroblasts, vessels within the scar tissue exhibited MMP-2 positivity (Figure 1d). To corroborate MMP-2 proteolytic activity with expression levels, results obtained by immunohistochemistry were compared with in situ zymograms. Although basal levels of MMP-2 protein expression were detected in healthy skin, proteolytic activity was nearly absent in in situ zymograms (Figure 2a and c). After injury, gelatinolytic activity could be visualized (translucent areas in Figure 2b). Semiquantitative
a
% MMP-2 positive cells/0.01 mm2
c
analysis of in situ zymography revealed enhanced MMP proteolytic activity at all time points in healing wounds. No gelatinolytic activity was detected when MMPs were blocked by a synthetic inhibitor (Figure 2d). In summary, MMP-2 protein expression and proteolytic activity are upregulated in healing wounds. MMP-2 transcription is induced via regulatory sequences 1686/ þ 423 in healing wounds
It is known that MMP-2 protein synthesis is regulated at the level of transcription as well as translation (Corcoran et al., 1996). Distinct regulatory elements within the MMP-2 gene promoter have been mapped previously, as outlined in the Introduction. In vitro, these regulatory elements confer cellspecific gene transcription. Transcriptional activity conferred by sequences 1686/ þ 423 was assessed in F8 animals (Figure 3a). Control-staining was performed by omitting the primary antibody (Figure 3b). Figure 3c illustrates dermal MMP-2-reporter gene expression in F8 mice 7 days after injury: Within the scar tissue, b-galactosidase-reporter gene expression was significantly enhanced. In healthy skin, b-galactosidase staining was restricted to the epidermal layer and hair follicles. The reporter-gene expression driven by MMP-2 regulatory sequences 1686/ þ 413 was upregulated over time in F8 mice (Figure 4a). Furthermore, b-galactosidase staining was obviously enhanced in F8 animals when compared with F8del animals with selectively deleted sequences 1686/ þ 1241 (Figure 4a and b). These differences in b-galactosidase expression patterns could be observed at all time points. The decreased reporter-gene expression in F8del mice indicates that RE-1 acts as an
b
50 m
60
d
50 40 30 20 10 0
Skin
Day 7
Day 21
Day 90 25 m
Figure 1. MMP-2 protein expression is enhanced in healing wounds. Immunohistochemistry for MMP-2 protein was performed and analyzed quantitatively for time points 7, 21, and 90 days. (a) MMP-2 protein expression in healthy skin is detectable within the epidermal layer, hair follicles, and in several cells interspersed in the subepidermal layer. Morphologically, these cells correspond to macrophages and fibroblasts. (b) MMP-2 protein expression is strongly enhanced 7 days after full dermal-skin incision. Bar ¼ 50 mm. (c) Morphometric analyses revealed significantly enhanced MMP-2 protein expression at all time points in healing wounds when compared with healthy skin. Tissue areas of 0.01 mm2 were analyzed in F8 and F8del transgenic mice (n ¼ 12 at each time point). Data are represented as means 7SEM. Differences between both strains were not detected (data not presented). (d) MMP-2 expression in vessel walls. Section of (b) is presented.
www.jidonline.org 1763
P Lynen Jansen et al. Regulation of MMP-2 Gene Transcription in Dermal Wounds
a
b
c
d Strong
Score
3
Moderate
2
Weak
1
Skin
0
Day 7
Day 21
Day 90
50 m
Figure 2. MMP proteolytic activity is enhanced in healing wounds. In situ zymography was performed and analyzed quantitatively for 7, 21, and 90 days. Translucent areas correspond to gelatinolytic activity. (a) Absence of proteolytic activity in healthy skin. (b) Areas of proteolytic activity 7 days after dermal incision, indicated by black arrows. (c) Semiquantitative analysis of in situ zymography reveals enhanced MMP proteolytic activity at all time points in healing wounds when compared with healthy skin. Tissue areas were analyzed in F8 and F8del transgenic mice and summarized (n ¼ 12 at each time point). Data are represented as means 7SEM. Differences between both strains were not detected (data not presented). (d) Control experiments with MMP-inhibitor 1. 10-phenanthroline were performed with specimen on day 7 after dermal incision. Bar ¼ 50 mm.
a
b
F8 + 423
−1686
Control staining
Lacz
Response element 1
c
Skin
Scar
Skin
200 m
Figure 3. MMP-2 transcription is induced via regulatory sequences 1686/ þ 423 in healing wounds. Immunohistochemistry for b-galactosidase was performed in F8 mice. (a) Schematic depiction of transgene introduced into F8 mice. LacZ-reporter gene is driven by MMP-2 regulatory sequences 1686/ þ 413 including the RE-1. (b) Control stainings were performed by omitting the primary anti-b-galactosidase antibody. (c) b-galactosidasereporter gene expression in F8 mice was significantly enhanced within the scar tissue. In contrast, b-galactosidase staining was restricted to the epidermal layer and hair follicles in healthy skin. A representative image taken with specimen from day 7 following dermal incision is shown. Bar ¼ 200 mm.
enhancer element of MMP-2 transcription in healing wounds in vivo. Localization of MMP-2 transcription
By means of b-galactosidase immunofluorescence, the distribution of MMP-2 transcription within the tissue specimens was further analyzed. As seen by immunohistochemistry, basal staining for b-galactosidase was detectable in hair follicles, within the epidermis and occasionally in fibroblasts 1764 Journal of Investigative Dermatology (2007), Volume 127
and residential macrophages in non-incised skin of F8 mice (Figure 5a). After injury, cells with intense b-galactosidase staining were evident (Figure 5b, white arrows). These cells were absent in F8del mice, whereas basal staining of b-galactosidase in hair follicles and within the epidermis still occurs (Figure 5c). Immunofluorescence costaining for b-galactosidase and CD68 or vWF revealed that MMP-2 regulatory sequence 1686/ þ 413, which includes the RE-1, is required for MMP-2 transcription in macrophages and vascular endothelial cells, respectively (Figure 6a and b). DISCUSSION In this report, we provide insights into the transcriptional regulation of MMP-2 expression during the course of dermalwound healing. We show that MMP-2 expression and proteolytic activity is upregulated in the subepidermal layer following skin injury. The results obtained by immunohistochemistry indicate an induction of MMP-2 protein expression over time, whereas MMP proteolytic activities were highest on day 7 and declined over time. It is likely that MMP proteolytic activity is subjected to regulatory interaction with tissue inhibitor of metalloproteinases or microtubule–MMPs. Ongoing MMP-2 protein synthesis provides a pool of latent enzyme ready for subsequent activation. To date, several therapeutic approaches have been taken to chemically block MMP activities at the protein level, but clinical trials with variably selective MMP inhibitors have been mostly unsuccessful. Our findings indicate that decreased transcriptional activity occurs following partial deletion of the RE-1. These data indicate that the RE-1 is an important cis-acting factor in injury related dermal-wound healing. In this regard, selective inactivation of MMP-2specific regulatory elements may be more promising. Such an
P Lynen Jansen et al. Regulation of MMP-2 Gene Transcription in Dermal Wounds
a
Day 7
Day 21
Day 90
F8 −1686 Response element 1
+ 423
Lacz
b F8del −1241
+ 423
Lacz 100 m
Figure 4. The MMP-2-regulatory sequences 1686/1241 are critically involved in conferring MMP-2 transcription in healing wounds. Immunohistochemical staining for b-galactosidase in F8 mice was compared with staining in F8del mice for time points 7, 21, and 90 days. (a) Reporter gene expression driven by MMP-2 regulatory sequences 1686/ þ 413 is induced over time in F8 mice. (b) The reporter gene expression is significantly reduced after deletion of regulatory sequences 1686/1241 at all time points in F8del mice. Bar ¼ 100 mm.
a
F8, skin (including RE-1)
-Galactosidase (red) DAPI (blue)
b
F8, scar (including RE-1)
c
F8del, scar
a
CD 68
-Galactosidase
DAPI
b
vWF
-Galactosidase
DAPI
(RE-1 deleted)
100 m
Figure 5. Localization of MMP-2 transcription is dependent on RE-1. Immunofluorescence staining for b-galactosidase (red) was performed. Nuclei were detected by DAPI staining (blue). (a) In healthy skin, staining for b-galactosidase was predominantly detected in hair follicles (F), within the epidermis (E) and occasionally in fibroblasts and residential macrophages within the dermis. (b) After dermal incision, areas of intense b-galactosidase expression within the dermis were detectable in F8 mice (white arrows). (c) Areas of intense b-galactosidase staining within the dermis were absent in F8del mice, whereas b-galactosidase staining was still detectable in hair follicles (F) and within the epidermis (E). Bar ¼ 100 mm.
approach was recently realized by Miyake et al. (2006), who successfully prevented the development of arterial aneurysm via blockage of MMP-2 transcription by NF-kB/ets1 oligonucleotide decoys in rabbits. The identification of RE-1 as a key regulatory element in MMP-2 transcription following dermal injury may provide a similar basis for the application of double-stranded oligodeoxynucleotides that are directed against one or even more specific cis-elements. Moreover, our data indicate that MMP-2 gene transcription was cell specifically regulated. We identified sequences 1686/1241 as critical for enhancement of MMP-2 transcription in macrophages and vascular endothelial cells in vivo. It is known that macrophages are strongly involved in MMP-2 expression and gene regulation at sites of inflammation. In vivo, Eerola et al. observed a direct relation between
50 m
Figure 6. MMP-2-regulatory sequences 1686/ þ 413, including RE-1, drive MMP-2 transcription in macophages and vascular endothelial cells. Immunofluorescence costaining of b-galactosidase and CD68 or vWF in F8 mice. (a) CD68 (red) positive macrophages exhibited b-galactosidase (green) costaining; (b) vWF (red) positive vascular endothelial cells exhibited b-galactosidase (green) costaining. Bar ¼ 50 mm.
MMP-2 gene activation and porcine posttransplant fibrotic reaction (Eerola et al., 2005). Modulation of MMP-2 expression might represent a novel approach for reorganizing cell–cell interactions with the aim to optimize wound healing or inflammation-associated diseases. Potential therapeutic targets are the transcription factors, for example Y-box protein-1 (YB-1), that bind to the RE-1 and cell-specifically trans-activates as well as transrepresses the MMP-2 promoter (Mertens et al., 1997). Interference with MMP-2 transcription may provide for cellspecific fine-tuning of the MMP-promoter transcriptional regulation. In conclusion, our findings highlight the complexity of MMP-2 gene regulation following dermal wounding, a key molecule for matrix remodelling. The established transgenic mice serve as useful models for further in vivo www.jidonline.org 1765
P Lynen Jansen et al. Regulation of MMP-2 Gene Transcription in Dermal Wounds
testing of ‘‘optimized’’ wound healing and neoangiogenesis with the aim to disrupt and/or enhance MMP-2 transcription. MATERIALS AND METHODS MMP-2/LacZ transgenic mice model The two mice strains used as animal models have been described in detail (Lee et al., 2005). In brief, mouse strain F8 harbor a aˆ-galactosidase-reporter gene (LacZ) under control of MMP-2 regulatory sequences 1686/ þ 423 that extend to the middle of the second exon. To determine the significance of the RE-1 that extends from 1282 to 1322 bp, F8del mice were utilized that harbor MMP2-regulatory sequence 1241/ þ 423, excluding the RE-1.
Study design and surgery The mice were fixed in the supine position. Full-thickness dermal incisions extending over 1.5 cm were performed 1 cm bilateral of the abdominal midline in 18 animals of each strain. The surgical procedure was performed under sterile conditions and general anesthesia by intramuscular administration of ketamine (ketamin 10%; Sanofi-Ceva, Du¨sseldorf, Germany) and xylazine (rompun 2%; Bayer, Leverkusen, Germany). After 7, 21, and 90 days of implantation, animals were killed by isoflurane asphyxation and decapitation. Tissue was harvested, split, and either fixed in 10% formalin for morphological analysis or quick-frozen and stored at 801C for activity assays. All animals received human care in accordance with the guidelines of the ‘‘Deutsche Tierschutzgesetz’’ (50.203.2-AC 46, 38/02) and the National Institutes of Health guidelines for the use of laboratory animals.
Histomorphological and immunohistochemical analyses Histological and immunohistochemical investigations were performed on paraffin-embedded 3-mm sections using peroxidaseconjugated, affinity-isolated Igs. All sections were routinely stained with hematoxylin and eosin and were processed at the same time to reduce internal staining variations or histomorphological studies. Immunohistochemistry was performed using the avidin–biotin complex and diaminobenzidine as chromogen. To detect cells with MMP-2-promoter activity, sections were incubated with monoclonal anti-b-galactosidase antibody (Europa, Cambridge, UK, 1:1700) followed by biotinylated rabbit anti-mouse IgG (Dako, Denmark, 1:300). Immunohistochemical staining was explored for MMP-2 (polyclonal rabbit, Biomol, Hamburg, 1:1000), a-smooth muscle actin (polyclonal rabbit, Abcam, Cambridge, UK, 1:300), CD68 (polyclonal rabbit, Santa Cruz, USA, 1:100), and vWF (polyclonal, Dako, Denmark, 1:400) followed by incubation with goat anti-rabbit IgG antibody (Dako, Denmark, 1:500). Coexpression of b-galactosidase with MMP-2, a-smooth muscle actin, CD68, and vWF was determined by immunofluorescence using goat anti-mouse, donkey anti-goat, and goat anti-rabbit secondary antibodies (Alexa Fluor 488, Molecular probes, Eugene, OR). Semiquantitative analyses of immunohistochemical staining (area 100 100 mm, 400-fold magnification) were performed with digital imaging analyzing software (Image-Pro Plus. Media Cybernetics, Silver Spring, MD). Control stainings were performed by omitting the primary antibodies.
Zymography Serial-frozen sections were placed on MMP in situ ZymoFilms composed of a 7-mm-thick layer of gelatin on a polyester base (Wako 1766 Journal of Investigative Dermatology (2007), Volume 127
Chemicals, Neuss, Germany) or MMP-PT in situ ZymoFilm, which contains the MMP inhibitor 1.10-phenanthroline. Films were incubated at 371C for 24 hours and stained with Biebrich Scarlet (Wako Chemical Industries, Japan). Gelatin digestion is visualized as transparency. Results obtained by comparison of both films indicate localization of MMP-specific proteolytic activity. Two blinded observers evaluated the gelatinolytic activity according to a scoring system (: negative; þ : weak; þ þ : moderate; þ þ þ : strong) in six independent tissue samples for each subgroup and time point.
Statistics The study was performed as balanced incomplete block design. Data were organized according to time, strain, and side. Differences between the groups were evaluated for statistical significance by analysis of variance describing the effect of strain, day, strain day, and side. Data were described as mean values 7SD. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft, DFG grant KL 1320/2-1 (UK, P.R.M.) and JA 1123/1-1 (P.L.J., UK, P.R.M., M.J.), SFB 542 project C4 (P.R.M.), IZKF-BIOMAT, RWTH-Aachen, Project no. NTV 41 and NIH grant DK039776 (D.H.L.).
REFERENCES Agren MS (1994) Gelatinase activity during wound healing. Br J Dermatol 131:634–40 Corcoran ML, Hewitt RE, Kleiner DE Jr, Stetler-Stevenson WG (1996) MMP-2: expression, activation and inhibition. Enzyme Protein 49:7–19 Eerola LM, Alho HS, Maasilta PK, Inkinen KA, Harjula AL, Litmanen SH et al. (2005) Matrix metalloproteinase induction in post-transplant obliterative bronchiolitis. J Heart Lung Transplant 24:426–32 Frisch SM, Morisaki JH (1990) Positive and negative transcriptional elements of the human type IV collagenase gene. Mol Cell Biol 10:6524–32 Grose R, Werner S (2004) Wound-healing studies in transgenic and knockout mice. Mol Biotechnol 28:147–66 Harendza S, Lovett DH, Stahl RAK (2000) The hematopoietic transcription factor PU.1 represses gelatinase A transcription in glomerular mesangial cells. J Biol Chem 275:19552–9 Harendza S, Pollock AS, Mertens PR, Lovett DH (1995) Tissue-specific enhancer-promoter interactions regulate high level constitutive expression of matrix metalloproteinase 2 by glomerular mesangial cells. J Biol Chem 270:18786–96 Ishikawa T, Nishigaki F, Miyata S, Hirayama Y, Minoura K, Imanishi J et al. (2005) Prevention of progressive joint destruction in collagen-induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR255031. Br J Pharmacol 144:133–43 Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, Itohara S (1998) Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res 58:1048–51 Jackson CJ, Xue M, Thompson P, Davey RA, Whitmont K, Smith S et al. (2005) Activated protein C prevents inflammation yet stimulates angiogenesis to promote cutaneous wound healing. Wound Repair Regen 13:284–94 Kato T, Kure T, Chang JH, Gabison EE, Itoh T, Itohara S et al. (2001a) Diminished corneal angiogenesis in gelatinase A-deficient mice. FEBS Lett 508:187–90 Kato T, Kure T, Chang JH, Gabison EE, Itoh T, Itohara S et al. (2001b) Diminished corneal angiogenesis in gelatinase A-deficient mice. FEBS Lett 508:187–90
P Lynen Jansen et al. Regulation of MMP-2 Gene Transcription in Dermal Wounds
Lee JG, Dahi S, Mahimkar R, Tulloch NL, Alfonso-Jaume MA, Lovett DH et al. (2005) Intronic regulation of matrix metalloproteinase-2 revealed by in vivo transcriptional analysis in ischemia. Proc Natl Acad Sci USA 102:16345–50 Mandal M, Mandal A, Das S, Chakraborti T, Chakraborti S (2003) Clinical implications of matrix metalloproteinases. Mol Cell Biochem 252:305–29 Marti HP, Lee L, Kashgarian M, Lovett DH (1994) Transforming growth factorbeta 1 stimulates glomerular mesangial cell synthesis of the 72-kd type IV collagenase. Am J Pathol 144:82–94 Mertens PR, Alfonso-Jaume MA, Steinmann K, Lovett DH (1998) A synergistic interaction of transcription factors AP2 and YB-1 regulates gelatinase A enhancer-dependent transcription. J Biol Chem 273:32957–65 Mertens PR, Alfonso-Jaume MA, Steinmann K, Lovett DH (1999) YB-1 regulation of the human and rat gelatinase A genes via similar enhancer elements. J Am Soc Nephrol 10:2480–7 Mertens PR, Harendza S, Pollock AS, Lovett DH (1997) Glomerular Mesangial Cell-specific Transactivation of Matrix Metalloproteinase 2a´Transcription Is Mediated by YB-1. J Biol Chem 272:22905–12 Mertens PR, Steinmann K, Alfonso-Jaume MA, En-Nia A, Sun Y, Lovett DH (2002) Combinatorial interactions of p53, AP2 and YB-1 with a single enhancer element regulate gelatinase A expression in neoplastic cells. J Biol Chem 277:24875–82
Miyake T, Aoki M, Nakashima H, Kawasaki T, Oishi M, Kataoka K et al. (2006) Prevention of abdominal aortic aneurysms by simultaneous inhibition of NF[kappa]B and ets using chimeric decoy oligonucleotides in a rabbit model. Gene Ther 13:695–704 Overall CM, Lopez-Otin C (2002) Strategies for MMP inhibition in cancer: innovations for the post-trial era. Nat Rev Cancer 2:657–72 Samolov B, Steen B, Seregard S, van dP I, Montan P, Kvanta A (2005) Delayed inflammation-associated corneal neovascularization in MMP-2-deficient mice. Exp Eye Res 80:159–66 Turck J, Pollock AS, Lee LK, Marti HP, Lovett DH (1996) Matrix metalloproteinase 2 (gelatinase A) regulates glomerular mesangial cell proliferation and differentiation. J Biol Chem 271:15074–83 Vaisanen A, Tuominen H, Kallioinen M, Turpeenniemi-Hujanen T (1996) Matrix metalloproteinase-2 (72 kD type IV collagenase) expression occurs in the early stage of human melanocytic tumour progression and may have prognostic value. J Pathol 180:283–9 Werner S, Grose R (2003) Regulation of wound healing by growth factors and cytokines. Physiol Rev 83:835–70 Wu M, Li YG (2006) The expression of CD40-CD40L and activities of matrix metalloproteinases in atherosclerotic rats. Mol Cell Biochem 282:141–6
www.jidonline.org 1767