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Overexpression of Membrane-Type Matrix Metalloproteinase-1 Gene Induces Mammary Gland Abnormalities and Adenocarcinoma in Transgenic Mice1

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Overexpression of Membrane-Type Matrix Metalloproteinase-1 Gene Induces Mammary Gland Abnormalities and Adenocarcinoma in Transgenic Mice1 [CANCER RESEARCH 61, 984–990, February 1, 2001] Overexpression of Membrane-type Matrix Metalloproteinase-1 Gene Induces Mammary Gland Abnormalities and Adenocarcinoma in Transgenic Mice1 Hye-Yeong Ha, Hyung-Bae Moon, Myoung-Soo Nam, Jeong-Woong Lee, Zae-Yoong Ryoo, Tae-Hoon Lee, Kyung-Kwang Lee, Byung-Jan So, Hiroshi Sato, Motoharu Seiki, and Dae-Yeul Yu2 Laboratory of Animal Developmental Biotechnology, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-333, Korea [H-Y. H., M-S. N., T-H. L., K-K. L., D-Y. Y.]; Research Institute of Medical Science, Catholic University of Korea, Seoul 137-040, Korea [J-W. L., Z-Y. R.]; Departments of Pathology [H-B. M.] and Surgery [B- J. S.], School of Medicine, Wonkwang University, Iksan 570-749, Korea; Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Kanazawa 920, Japan [H. S.]; and Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo 108-639, Japan [M. S.] ABSTRACT brane domains as MT-MMP-1 through -5, these enzymes have been proposed to be the master switches of ECM turnover based on the To investigate the role of membrane-type matrix metalloproteinase-1 purported ability of MT-MMPs to activate other MMPs such as (MT1-MMP) in mammary gland development and tumorigenesis, trans- proMMP-2 and MMP-13. ProMMP-2 and MMP-13 are degradative genic mice overexpressing MT1-MMP in mammary gland under the con- trol of the mouse mammary tumor virus long terminal repeat-promoter enzymes widely implicated in tumor invasion and metastasis (10, 16, 17). were generated. The mouse mammary tumor virus/MT1-MMP transgenic As do other MMPs, MT1-MMP has also been proposed to play mice displayed abnormalities in 82% of female mammary glands. The critical roles in both physiology and pathology by remodeling the abnormalities were verified as lymphocytic infiltration, fibrosis, hyper- ECM. MT1-MMP expression is particularly high in kidney during plasia, alveolar structure disruption, dysplasia, and adenocarcinoma. mouse embryogenesis and also in the adult human (12, 18). Recent Northern and reverse transcription-PCR analyses demonstrated that data indicate that MT1-MMP may also function as a fibrinolytic MT1-MMP mRNA was overexpressed in mammary glands exhibiting enzyme in the absence of plasmin and mediate pericellular proteolysis abnormalities. Western blot analysis and immunohistochemical studies in angiogenesis (19). Recently it was reported that MT1-MMP-defi- have revealed that the protein expression level was also increased in these cient mice develop dwarfism, osteopenia, arthritis, and connective glands. In addition, the ␤-casein gene as a functional epithelial cell marker tissue disease because of inadequate collagen turnover (20). MT1- was poorly expressed in the mammary glands of transgenic mice exhib- iting abnormalities. Gelatin zymography showed significantly increased MMP is also overexpressed in various tumor tissues, including human MMP-2 activation in these mammary glands. These results showed that colon, breast, and head and neck carcinoma (10, 21–24). Although overexpression of MT1-MMP induced remodeling of the extracellular MT1-MMP expression has been proved in numerous tumors, the roles matrix and tumor formation in the mammary glands of transgenic mice. assigned to MT1-MMP in tumorigenesis and tumor progression are Therefore, we suggest that overexpression of MT1-MMP may play a key relatively poorly understood. In the present study, we generated role in development and tumorigenesis in mammary glands. MMTV/MT1-MMP transgenic mice and examined premalignant ab- normalities and adenocarcinoma in mammary glands. The results INTRODUCTION suggest that overexpression of MT1-MMP may play a key role in development and tumorigenesis in mammary glands. MMPs,3 which degrade the various components of ECM, play critical roles in the tissue remodeling of multicellular organisms as well as in tumor invasion (1–4). MMPs may play a role in any one of MATERIALS AND METHODS multiple critical events in tumor evolution, including tumorigenesis, tumor growth, angiogenesis, generation of reactive stroma, and tumor Generation of MMTV/MT1-MMP Transgenic Mice. To generate a vec- cell invasion and metastasis (5). For example, the lack of MMP-7 in tor pmMT1, a 1.8-kb mouse MT1-MMP cDNA of full length for the coding mice showed a reduction in intestinal tumorigenesis (6), and its sequence was ligated into the SalI and XhoI sites of the mammalian expression vector pMAM-neo (Clonetech, Palo Alto, CA; Ref. 25). A HindIII DNA overexpression in mammary tissue accelerates mammary tumor for- fragment (4.4 kb) containing MMTV-LTR, MT1-MMP cDNA, and SV40 mation in mice carrying the MMTV/ErbB-2 transgene (7). In addition, polyadenylation sequences was microinjected into the pronuclei of fertilized MMP-2-defective mice showed reduced angiogenesis and tumor pro- mouse eggs obtained from C57BL/6 ϫ DBA F1 hybrid females as described gression (8). MMP-11 knockout mice showed reduced tumorigenesis (Ref. 26; Fig. 1). The DNA-injected eggs were transferred to pseudopregnant in response to chemical mutagenesis (9). ICR female mice. Transgenic mice were identified by PCR analysis of the Whereas the majority of the MMPs are secreted as soluble enzymes genomic DNA using primers specific to mMT1-injection DNA. The oligonu- into the extracellular milieu, a subset of MMPs have been identified cleotides used for the amplification were a forward primer 5Ј-ACA-AGA- in recent years to contain additional sequences capable of anchoring GCG-CAA-CGG-ACT-CA-3Ј complementary to MMTV LTR gene sequences on plasma membrane (10–15). Named after the putative transmem- and 5Ј-ACG-GTG-TAA-GCT-CCG-GTA-3Ј specific to the MT1-MMP gene. Histological and Immunohistochemical Stain. Mammary tissues were obtained from wild-type and transgenic mice at various stages of development. Received 11/17/99; accepted 11/20/00. Tissues were fixed in neutral buffered 10% formalin overnight and embedded The costs of publication of this article were defrayed in part by the payment of page ␮ charges. This article must therefore be hereby marked advertisement in accordance with in paraffin, sectioned at 4 m, and stained with H&E. For immunohistochem- 18 U.S.C. Section 1734 solely to indicate this fact. ical staining, the 4-␮m paraffin-embedded sections were prepared on the 1 This work was supported by grants NB0540 and NB0870 from the Ministry of Probe-on Plus slides (Fisher, Pittsburgh, PA) and deparaffinized by xylene. Science and Technology of Korea. Next, tissue sections were rehydrated in PBS solution, and then the slides were 2 To whom requests for reprints should be addressed, at Laboratory of Animal Developmental Biotechnology, Korea Research Institute of Bioscience and Biotechnol- blocked in 3% hydrogen peroxide for 10 s. The slides were washed twice in ogy, Taejon 305-333, Korea. Phone: 82-42-860-4422; Fax: 82-42-860-4608; E-mail: Immuno/DNA buffer solution (Research Genetics, Huntsville, AL) and then [email protected]. incubated in protein blocker solution (Research Genetics) for 3 min. The 3 The abbreviations used are: MMP, matrix metalloproteinase; MT, membrane-type; sections were incubated at 4°C overnight with the monoclonal antibody, ECM, extracellular matrix; RT-PCR, reverse transcription-PCR; MMTV LTR, mouse mammary tumor virus long terminal repeat; GAPDH, glyceraldehyde-3-phosphate dehy- 113-5B7 against MT1-MMP (10), and incubated with the universal secondary drogenase. antibody (Research Genetics). The sections were incubated with diaminoben- 984 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2001 American Association for Cancer Research. MAMMARY ADENOCARCINOMA IN MT1-MMP TRANSGENIC MICE water washes, and incubated overnight in 50 mM Tris-HCl (pH 7.5) containing 10 mM CaCl2, 0.5 M NaCl, and 0.02% NaN3 at 37°C. After incubation, the gel was stained with 0.25% Coomassie Blue R-250 and destained with 10% methanol and 10% acetic acid. Proteolytic bands appeared clear on blue- stained background. RESULTS Generation of Transgenic Mice. Transgenic mice were generated by microinjecting a 4.4-kb HindIII DNA fragment containing the mouse MT1-MMP cDNA under the transcriptional control of the MMTV LTR promoter. Transgenic mice were identified by PCR analysis, and two female mice and one male founder mouse were obtained (Fig. 1). Transgenic mouse lines were established by mating transgenic founder mice to C57BL/6 mice. All of the founders were fertile and capable of transmitting the transgene to progeny. Expres- Fig. 1. Generation of MMTV/MT1-MMP transgenic mice. A, structure of pmMT1 sion of the MMTV/MT1-MMP transgene in various stages of mam- vector. The pmMT1 vector was constructed by inserting 1.8 kb of mouse MT1-MMP mary gland development was examined by RT-PCR. The expression cDNA into the pMAMneo vector (see “Materials and Methods”). The 4.4 kb of the HindIII fragment of MMTV-LTR, mouse MT1-MMP cDNA, and SV40 polyadenylation of transgene mRNA was readily detectable throughout all of the site [SV40 poly(A)] was microinjected into fertilized eggs. B, identification of MMTV/ stages (data not shown). Two lines, designated nos. 4 and 11, were MT1-MMP transgenic mice by PCR analysis. The primer sequences were described in “Materials and Methods.” The expected fragment (440 bp) was indicated in A. Founders selected for the additional experiments because the female founder no. 4 and 11 were female; no. 12 was male. showed poor lactation after the second and sixth parturitions, respec- tively. MT1-MMP Overexpression Induces Abnormalities in Trans- zidine for 10 min and washed with Immuno/DNA (Research Genetics). May- er’s hematoxylin (Research Genetics) was used as counterstain, and the slides genic Mammary Glands. To determine whether the expression of were mounted with universal mount (Research Genetics). the MT1-MMP transgene affected morphology of the transgenic mam- Northern Blot Analysis.
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