Prostate Cancer-Associated Membrane Type 1-Matrix Metalloproteinase
2007; Elsevier BV; Volume: 170; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2007.060720
ISSN1525-2191
AutoresR. Daniel Bonfil, Zhong Dong, J. Carlos Trindade Filho, Aaron Sabbota, Pamela Osenkowski, Sanaa Nabha, Hamilto Yamamoto, Sreenivasa R. Chinni, Huiren Zhao, Shahriar Mobashery, Robert L. Vessella, Rafael Fridman, Michael L. Cher,
Tópico(s)Bone Metabolism and Diseases
ResumoMembrane type 1-matrix metalloproteinase (MT1-MMP) is a major mediator of collagen I degradation. In human samples, we show that prostate cancer cells in skeletal metastases consistently express abundant MT1-MMP protein. Because prostate cancer bone metastasis requires remodeling of the collagen-rich bone matrix, we investigated the role of cancer cell-derived MT1-MMP in an experimental model of tumor-bone interaction. MT1-MMP-deficient LNCaP human prostate cancer cells were stably transfected with human wild-type MT1-MMP (MT1wt). Furthermore, endogenous MT1-MMP was down-regulated by small interfering RNA in DU145 human prostate cancer cells. Intratibial tumor injection in severe combined immunodeficient mice was used to simulate intraosseous growth of metastatic tumors. LNCaP-MT1wt cells produced larger osseous tumors than Neo control cells and induced osteolysis, whereas DU145 MT1-MMP-silenced transfectants induced osteogenic changes. In vitro assays showed that MT1wt overexpression enhanced collagen I degradation, whereas MT1-MMP-silencing did the opposite, suggesting that tumor-derived MT1-MMP may contribute directly to bone remodeling. LNCaP-MT1wt-derived conditioned medium stimulated in vitro multinucleated osteoclast formation. This effect was inhibited by osteoprotegerin, a decoy receptor for receptor activator of nuclear factor κB ligand, and by 4-[4-(methanesulfonamido) phenoxy] phenylsulfonyl methylthiirane, an MT1-MMP inhibitor. Our findings are consistent with the hypothesis that prostate cancer-associated MT1-MMP plays a direct and/or indirect role in bone matrix degradation, thus favoring intraosseous tumor expansion. Membrane type 1-matrix metalloproteinase (MT1-MMP) is a major mediator of collagen I degradation. In human samples, we show that prostate cancer cells in skeletal metastases consistently express abundant MT1-MMP protein. Because prostate cancer bone metastasis requires remodeling of the collagen-rich bone matrix, we investigated the role of cancer cell-derived MT1-MMP in an experimental model of tumor-bone interaction. MT1-MMP-deficient LNCaP human prostate cancer cells were stably transfected with human wild-type MT1-MMP (MT1wt). Furthermore, endogenous MT1-MMP was down-regulated by small interfering RNA in DU145 human prostate cancer cells. Intratibial tumor injection in severe combined immunodeficient mice was used to simulate intraosseous growth of metastatic tumors. LNCaP-MT1wt cells produced larger osseous tumors than Neo control cells and induced osteolysis, whereas DU145 MT1-MMP-silenced transfectants induced osteogenic changes. In vitro assays showed that MT1wt overexpression enhanced collagen I degradation, whereas MT1-MMP-silencing did the opposite, suggesting that tumor-derived MT1-MMP may contribute directly to bone remodeling. LNCaP-MT1wt-derived conditioned medium stimulated in vitro multinucleated osteoclast formation. This effect was inhibited by osteoprotegerin, a decoy receptor for receptor activator of nuclear factor κB ligand, and by 4-[4-(methanesulfonamido) phenoxy] phenylsulfonyl methylthiirane, an MT1-MMP inhibitor. Our findings are consistent with the hypothesis that prostate cancer-associated MT1-MMP plays a direct and/or indirect role in bone matrix degradation, thus favoring intraosseous tumor expansion. A major clinical complication of prostate cancer patients is the development of skeletal metastasis. Intraosseous growth of cancer requires the action of proteolytic enzymes to facilitate remodeling of the bone matrix, promote angiogenesis, and increase the availability of local growth factors. Matrix metalloproteinases (MMPs) have been shown to play a pivotal role in the pathogenesis of osteolytic bone metastases.1Yoneda T Sasaki A Dunstan C Williams PJ Bauss F De Clerck YA Mundy GR Inhibition of osteolytic bone metastasis of breast cancer by combined treatment with the bisphosphonate ibandronate and tissue inhibitor of the matrix metalloproteinase-2.J Clin Invest. 1997; 99: 2509-2517Crossref PubMed Scopus (226) Google Scholar, 2Lee J Weber M Mejia S Bone E Watson P Orr W A matrix metalloproteinase inhibitor, batimastat, retards the development of osteolytic bone metastases by MDA-MB-231 human breast cancer cells in Balb C nu/nu mice.Eur J Cancer. 2001; 37: 106-113Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Previously, we and others showed that systemic inhibition of MMP activity reduces tumor cell proliferation, disrupts osteoclast recruitment, and suppresses bone degradation in animal models of bone metastasis.2Lee J Weber M Mejia S Bone E Watson P Orr W A matrix metalloproteinase inhibitor, batimastat, retards the development of osteolytic bone metastases by MDA-MB-231 human breast cancer cells in Balb C nu/nu mice.Eur J Cancer. 2001; 37: 106-113Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 3Nemeth JA Yousif R Herzog M Che M Upadhyay J Shekarriz B Bhagat S Mullins C Fridman R Cher ML Matrix metalloproteinase activity, bone matrix turnover, and tumor cell proliferation in prostate cancer bone metastasis.J Natl Cancer Inst. 2002; 94: 17-25Crossref PubMed Scopus (185) Google Scholar, 4Winding B NicAmhlaoibh R Misander H Hoegh-Andersen P Andersen TL Holst-Hansen C Heegaard AM Foged NT Brunner N Delaisse JM Synthetic matrix metalloproteinase inhibitors inhibit growth of established breast cancer osteolytic lesions and prolong survival in mice.Clin Cancer Res. 2002; 8: 1932-1939PubMed Google Scholar, 5Weber MH Lee J Orr FW The effect of Neovastat (AE-941) on an experimental metastatic bone tumor model.Int J Oncol. 2002; 20: 299-303PubMed Google Scholar Recently, we reported up-regulation of MMP-9 activity shortly after the bone microenvironment was colonized by prostate cancer cells.6Dong Z Bonfil RD Chinni S Deng X Trindade Filho JC Bernardo M Vaishampayan U Che M Sloane BF Sheng S Fridman R Cher ML Matrix metalloproteinase activity and osteoclasts in experimental prostate cancer bone metastasis tissue.Am J Pathol. 2005; 166: 1173-1186Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Membrane type 1-matrix metalloproteinase (MT1-MMP) is a membrane-anchored protease that has the capacity to activate pro-MMP-2 on the cell surface7Itoh Y Takamura A Ito N Maru Y Sato H Suenaga N Aoki T Seiki M Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion.EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (335) Google Scholar and to promote tumor growth and angiogenesis.8Sounni NE Devy L Hajitou A Frankenne F Munaut C Gilles C Deroanne C Thompson EW Foidart JM Noel A MT1-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression.FASEB J. 2002; 16: 555-564Crossref PubMed Scopus (234) Google Scholar, 9Chun TH Sabeh F Ota I Murphy H McDonagh KT Holmbeck K Birkedal-Hansen H Allen ED Weiss SJ MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix.J Cell Biol. 2004; 167: 757-767Crossref PubMed Scopus (274) Google Scholar MT1-MMP degrades several extracellular matrix components including type I collagen,10Ohuchi E Imai K Fujii Y Sato H Seiki M Okada Y Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules.J Biol Chem. 1997; 272: 2446-2451Crossref PubMed Scopus (824) Google Scholar the most abundant matrix protein in bone.11Eyre DR Collagen: molecular diversity in the body's protein scaffold.Science. 1980; 207: 1315-1322Crossref PubMed Scopus (337) Google Scholar In fact, mice deficient in MT1-MMP display a prominent skeletal phenotype due to abnormalities in bone remodeling.12Holmbeck K Bianco P Pidoux I Inoue S Billinghurst RC Wu W Chrysovergis K Yamada S Birkedal-Hansen H Poole AR The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone.J Cell Sci. 2005; 118: 147-156Crossref PubMed Scopus (173) Google Scholar, 13Zhou Z Apte SS Soininen R Cao R Baaklini GY Rauser RW Wang J Cao Y Tryggvason K Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I.Proc Natl Acad Sci USA. 2000; 97: 4052-4057Crossref PubMed Scopus (676) Google Scholar Previously, in human prostate tissues, we showed that MT1-MMP is expressed in the basal cells of benign glands, in the secretory cells of prostatic intraepithelial neoplasia, and in some invasive prostate adenocarcinoma glands.14Upadhyay J Shekarriz B Nemeth JA Dong Z Cummings GD Fridman R Sakr W Grignon DJ Cher ML Membrane type 1-matrix metalloproteinase (MT1-MMP) and MMP-2 immunolocalization in human prostate: change in cellular localization associated with high-grade prostatic intraepithelial neoplasia.Clin Cancer Res. 1999; 5: 4105-4110PubMed Google Scholar Together, these studies suggest that tumor-associated MT1-MMP activity may promote prostate cancer progression and metastasis. In this study, we present the first description of MT1-MMP expression in human prostate cancer bone metastasis. We demonstrate that overexpression of MT1-MMP in prostate cancer cells promotes intraosseous tumor growth and an osteolytic response in an in vivo model, whereas down-regulation of MT1-MMP generates virtually the opposite results. Tumor-derived MT1-MMP may contribute to tumor growth and bone remodeling directly by degradation of bone matrix and indirectly by shedding of soluble receptor activator of nuclear factor κB ligand (sRANKL), which can promote osteoclast recruitment and differentiation. The human prostate cancer cell lines LNCaP and DU145 (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium and Dulbecco's modified Eagle's medium, respectively, both supplemented with 10% fetal bovine serum, at 37°C and 5% CO2. The human full-length wild-type MT1-MMP cDNA was constructed into the pcDNA 3.1/myc-His (−) expression vector (Invitrogen, Carlsbad, CA) using appropriate restriction sites. LNCaP cells were stably transfected with pcDNA 3.1 vector containing full-length wild-type (MT1wt) MT1-MMP using Effectene Transfection Reagent (Qiagen, Valencia, CA), based on the manufacturer's instructions. Control LNCaP cells (Neo) were transfected with the pcDNA 3.1 vector without MT1-MMP DNA insert. Stable cell lines (pooled populations) were selected and maintained in culture medium supplemented with G-418 (Invitrogen). MT1-MMP small interfering RNA (siRNA) was designed using siRNA Target Finder (Ambion, Austin, TX; http://www.ambion.com/techlib/misc/siRNA_finder.html), and the selected target sequence was 5′-AAGTCTTCACTTACTTCTACA-3′. The siRNA targeting the sequence was synthesized using a Silencer siRNA Construction kit (Ambion), and its silencing effectiveness was tested by treatment of the MT1-MMP-expressing DU145 human prostate cancer cells with the synthesized MT1-MMP siRNA. An MT1-MMP siRNA-expressing DNA insert was constructed into the pSilencer Hygro siRNA Expression Vector (Ambion). The vector containing the MT1-MMP siRNA (MT1si)-expressing insert was amplified, purified, and sequenced. DU145 cells were transfected with either the MT1si-expressing vector or a vector expressing a scrambled siRNA (scr-si) that did not affect MT1-MMP expression. The transfected cells were selected by hygromycin treatment, and the resistant cells were further cloned. The murine monocytic cell line RAW 264.715Raschke WC Baird S Ralph P Nakoinz I Functional macrophage cell lines transformed by Abelson leukemia virus.Cell. 1978; 15: 261-267Abstract Full Text PDF PubMed Scopus (629) Google Scholar (American Type Culture Collection) was cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium with 4 mmol/L l-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose and supplemented with 10% fetal bovine serum. Total RNA was extracted from LNCaP and DU145 transfectant cells using TRIzol reagent (Invitrogen), according to the manufacturer's instructions. MT1-MMP mRNA was amplified with the primers of MT1-MMP (forward, 5′-CGCTACGCCATCCAGGGTCTCAAA-3′; and reverse, 5′-CGGTCATCATCGGGCAGCACAAAA-3′). RANKL primers used were 5′-TCCCATCTGGTTCCATAAA-3′ (forward) and 5′-ATCCAGTAAGGAGGGGTTGG-3′ (reverse). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also amplified as an internal control to compare relative expression of the target genes among different samples. Polymerase chain reaction (PCR) products were run on a 1% agarose gel and visualized by ethidium bromide staining. Bone metastasis tissue samples (n = 20) were obtained from rapid autopsies of prostate cancer patients.16Roudier MP Vesselle H True LD Higano CS Ott SM King SH Vessella RL Bone histology at autopsy and matched bone scintigraphy findings in patients with hormone refractory prostate cancer: the effect of bisphosphonate therapy on bone scintigraphy results.Clin Exp Metastasis. 2003; 20: 171-180Crossref PubMed Scopus (81) Google Scholar These patients had androgen-independent disease, as previously reported.17Lai JS Brown LG True LD Hawley SJ Etzioni RB Higano CS Ho SM Vessella RL Corey E Metastases of prostate cancer express estrogen receptor-beta.Urology. 2004; 64: 814-820Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Aside from two patients with no associated bone change, all samples revealed a diffuse osteoblastic reaction to prostate cancer cells.16Roudier MP Vesselle H True LD Higano CS Ott SM King SH Vessella RL Bone histology at autopsy and matched bone scintigraphy findings in patients with hormone refractory prostate cancer: the effect of bisphosphonate therapy on bone scintigraphy results.Clin Exp Metastasis. 2003; 20: 171-180Crossref PubMed Scopus (81) Google Scholar Five-micrometer paraffin sections were immunostained using a rabbit polyclonal antibody against the catalytic domain of MT1-MMP (Spring Bioscience, Fremont, CA) and the Vectastain Elite ABC peroxidase kit (Vector Laboratories, Burlingame, CA), following manufacturer's instructions. LNCaP-Neo and LNCaP-MT1wt cells cultured to 80% confluence were washed twice with phosphate-buffered saline and then lysed in Nonidet P-40 lysis buffer (25 mmol/L Tris, pH 7.5, 100 nmol/L NaCl, and 1% Nonidet P-40) in the presence of a protease inhibitor cocktail not containing ethylenediamine tetraacetic acid (Roche Applied Science, Indianapolis, IN). Protein concentrations were determined using the bicinchoninic acid method (Pierce Chemical Company, Rockford, IL). To detect MT1-MMP, equal amounts of whole-cell lysates (50 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions followed by immunoblotting using the LEM-2/15 monoclonal antibody to the catalytic domain of human MT1-MMP (1:2000) (kindly provided by Dr. A. Arroyo, Hospital de la Princesa, Madrid, Spain). After washing, the membranes were incubated with an anti-mouse secondary antibody conjugated with horseradish peroxidase (1:3000; Cell Signaling Technology, Danvers, MA). An MT1-MMP enriched membrane preparation from nonmalignant monkey kidney epithelial BS-C-1 cells was electrophoresed as a positive control.18Hernandez-Barrantes S Toth M Bernardo MM Yurkova M Gervasi DC Raz Y Sang QA Fridman R Binding of active (57 kDa) membrane type 1-matrix metalloproteinase (MT1-MMP) to tissue inhibitor of metalloproteinase (TIMP)-2 regulates MT1-MMP processing and pro-MMP-2 activation.J Biol Chem. 2000; 275: 12080-12089Crossref PubMed Scopus (272) Google Scholar The blots were stripped and re-probed with an antibody to β-actin (Sigma-Aldrich, St. Louis, MO). Membranes were subjected to enhanced chemiluminescence detection (Pierce), according to manufacturer's instructions. MT1-MMP activity in cell extracts was measured using the Biotrak MMP-14 activity assay (GE Healthcare, Little Chalfont, Buckinghamshire, UK), as described by the manufacturer. For pro-MMP-2 activation, LNCaP transfectant cells were incubated (2 hours, 37°C) with serum-free medium supplemented with 2 nmol/L recombinant pro-MMP-2, which was produced in HeLa S3 cells infected with the appropriate recombinant vaccinia viruses.19Bernardo MM Fridman R TIMP-2 (tissue inhibitor of metalloproteinase-2) regulates MMP-2 (matrix metalloproteinase-2) activity in the extracellular environment after pro-MMP-2 activation by MT1 (membrane type 1)-MMP.Biochem J. 2003; 374: 739-745Crossref PubMed Scopus (150) Google Scholar Cell lysates were then obtained and subjected to gelatin zymography as described previously.6Dong Z Bonfil RD Chinni S Deng X Trindade Filho JC Bernardo M Vaishampayan U Che M Sloane BF Sheng S Fridman R Cher ML Matrix metalloproteinase activity and osteoclasts in experimental prostate cancer bone metastasis tissue.Am J Pathol. 2005; 166: 1173-1186Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Alternatively, recombinant pro-MMP-2 was incubated either with an equal amount of cell lysates prepared from DU145 clones or with the same number of live DU145 cells in serum-free Dulbecco's modified Eagle's medium at 37°C for 16 hours. The lysates and culture media were collected and subjected to gelatin zymography. Rat type I collagen (5 mg/ml solution; Trevigen, Gaithersburg, MD) was diluted to 2 mg/ml using a solution composed of 10× RPMI 1640 medium culture medium, distilled water, and 1 N NaOH (4:1:4.8:0.2 ratio), at 4°C. LNCaP transfectant cells were suspended in the solution (1 × 104 cells/ml), and 1 ml of the cell suspension was dispensed per well in six-well plates. Each mixture was allowed to polymerize at 37°C for 1 hour. Growth culture medium (3 ml/well) was then added on top of the gels. After 18 days, the gels were dissolved with 2 mg/ml bacterial collagenase (Worthington Biochemical Corporation, Lakewood, NJ), and cells were counted with a hemocytometer. For DU145 clones, the assay was performed similarly, except that the collagen gels with cells were dissolved after 6 days. Five-week-old male C.B.-17 severe combined immunodeficient (SCID) mice (Taconic Farms, Germantown, NY) were randomly divided into six groups of 9 to 10 animals. For LNCaP transfectants, 2 × 105 cells in 10 μl of serum-free medium were injected into the proximal end of tibiae 4 to 5 mm down the diaphysis using calibrated Microliter syringes (Hamilton Syringe Co., Reno, NV) and 27-gauge needles under anesthesia. For DU145 transfectants (DU145-scr-si clone and DU145-MT1si clones 1, 5, and 8), 1 × 105cells in 10 μl of medium were injected the same way. Lukens bone wax (Surgical Specialties Co., Reading, PA) was applied over the site of injection to prevent cell leakage. Prostatic specific antigen levels were determined in plasma of mice intratibially injected with LNCaP using an enzyme-linked immunosorbent assay kit (Anogen, Mississauga, ON, Canada), according to the manufacturer's instructions. For subcutaneous growth, 5 × 106 LNCaP transfectant cells were inoculated in male SCID mice (n = 5). X-rays of whole mice were obtained every 2 to 3 weeks with a mammography unit. All procedures were done in compliance with the Animal Investigation Committee of Wayne State University and National Institutes of Health guidelines. Tibiae were fixed and decalcified in formic acid/formaldehyde (Cal-Rite; Richard-Allan Scientific, Kalamazoo, MI). Five-micrometer-thick longitudinal sections were stained with hematoxylin and eosin or immunostained for cytokeratin, as described previously.6Dong Z Bonfil RD Chinni S Deng X Trindade Filho JC Bernardo M Vaishampayan U Che M Sloane BF Sheng S Fridman R Cher ML Matrix metalloproteinase activity and osteoclasts in experimental prostate cancer bone metastasis tissue.Am J Pathol. 2005; 166: 1173-1186Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Tibiae with no evident intraosseous tumor, as confirmed by immunohistochemistry for cytokeratin, were excluded from the analysis. Digital photomicrographs were captured under ×5 magnification using a Zeiss Axioplan 2 microscope (Zeiss, Göttingen, Germany) equipped with a software-controlled (Axiovision; Zeiss) digital camera. All ×5-microscopic fields found in each longitudinal section cut through the middle part of the tibiae were analyzed. The jpeg images obtained were then merged to get a panoramic view of the whole sagittal section of the tibia. The percentage occupied by tumor and trabecular and cortical bone in the histological section of the entire tibia was calculated by the software based on the measurement of the corresponding areas in pixels.2Lee J Weber M Mejia S Bone E Watson P Orr W A matrix metalloproteinase inhibitor, batimastat, retards the development of osteolytic bone metastases by MDA-MB-231 human breast cancer cells in Balb C nu/nu mice.Eur J Cancer. 2001; 37: 106-113Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar In certain cases, only bone regions containing tumor cells were selected to calculate the percentage of tumor-associated trabecular bone tissues. These regions were defined by left and right edges of intraosseous tumor nests, respectively, whereas top and bottom sides were the outer edges of cortical bone included in the segment. Several tumor-associated regions can be found in a whole-bone tissue slide. In all cases, only tibiae that showed intraosseous tumor growth were selected for histomorphometrical analysis. The ability of LNCaP-MT1wt and LNCaP-Neo cells to degrade type I collagen was measured using their lysates (prepared as explained above) with the ENzChek Collagenase Assay kit (Molecular Probes, Eugene, OR), according to the manufacturer's instructions. Mouse bone marrow primary cultures or Raw 264.7 cells were used to investigate the osteoclastogenic effect of factors shed by the LNCaP transfectants. In the first case, bone marrow cells isolated from femora and tibiae from male mice were cultured in α-minimal essential medium containing 10% fetal bovine serum and macrophage colony-stimulating factor (100 ng/ml; R&D Systems, Minneapolis, MN) on LUX coverslips (Miles Scientific, Division of Miles Laboratories, Inc., Naperville, IL) in 24-well culture plates.20Kobayashi K Takahashi N Jimi E Udagawa N Takami M Kotake S Nakagawa N Kinosaki M Yamaguchi K Shima N Yasuda H Morinaga T Higashio K Martin TJ Suda T Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction.J Exp Med. 2000; 191: 275-286Crossref PubMed Scopus (1056) Google Scholar Three days later, nonadherent cells were removed, and the cultures were exposed to protein-normalized 48-hour conditioned medium (40%; conditioned medium volume/total culture volume) from either LNCaP-Neo or LNCaP-MT1wt cells, supplemented with fetal bovine serum to reach a final concentration of 0.5%. Seventy-two hours later, the cells were fixed and stained with Diff-Quik kit (Dade Behring, Newark, DE), according to the manufacturer's instructions. As for Raw 264.7 cells, 2 × 104 cells in complete culture medium were seeded in each well on top of LUX coverslips and incubated at 37°C for 24 hours. The coverslips were washed once and then exposed to conditioned medium from LNCaP-Neo or LNCaP-MT1wt, as described above. RANKL (50 ng/ml; PeproTech, Inc., Rocky Hill, NJ) was used as a positive control and 48-hour culture medium (40% final volume) as a negative control. Recombinant human osteoprotegerin (100 ng/ml; Leinco Technologies, St. Louis, MO) was used to block RANKL-induced differentiation of preosteoclasts into osteoclast-like cells. In a different experiment, to confirm the function of MT1-MMP on membrane-tethered RANKL shedding, conditioned media were obtained from LNCaP-Neo and LNCaP-MT1wt cells incubated for 48 hours with 10 μmol/L 4-[4-(methanesulfonamido) phenoxy] phenylsulfonyl methylthiirane (MIK-G2), an MT1-MMP and gelatinase inhibitor,21Ikejiri M Bernardo MM Bonfil RD Toth M Chang M Fridman R Mobashery S Potent mechanism-based inhibitors for matrix metalloproteinases.J Biol Chem. 2005; 280: 33992-34002Crossref PubMed Scopus (115) Google Scholar or the vehicle (culture medium supplemented with 0.3% polyethylene glycol and 0.1% dimethyl sulfoxide). LNCaP cells do not secrete gelatinases A and B (data not shown), and consequently, MIK-G2 was used in this experiment as a selective inhibitor for MT1-MMP in the LNCaP-MT1wt cells. In all of the wells in which Raw 264.7 cells were exposed to the different conditioned media, fetal bovine serum was added to reach a final concentration of 0.5%. Five days later, the cells were fixed and stained with Diff-Quik kit. Osteoclast-like differentiation was assessed in digital photomicrographs of either bone marrow primary cultures or Raw 264.7 cells captured under ×40 magnification in five fields at random using a Zeiss Axioplan 2 microscope (Zeiss). Tartrate-resistant acid phosphatase staining was performed to confirm histochemically osteoclast-like differentiation. Briefly, cells were fixed for 10 minutes with 4% paraformaldehyde in phosphate-buffered saline, washed three times with phosphate-buffered saline, and then stained for tartrate-resistant acid phosphatase using the Diagnostic Acid Phosphatase kit (Sigma) according to the manufacturer's instructions. All experiments were performed in triplicate. Data comparing differences between two groups were statistically analyzed using unpaired Student's t-test. Multiple comparisons were made using one-way analysis of variance with Tukey-Kramer post-testing. Differences were considered significant when P < 0.05. Paraffin sections of bone metastases obtained from 20 prostate cancer patient autopsies were immunostained for MT1-MMP. As shown in Figure 1, A and C–E, all of the specimens exhibited strong immunolocalization of MT1-MMP to prostate cancer cells. In areas of bone tissue without metastatic tumor, MT1-MMP immunoreactivity was also detected in endothelial cells, osteocytes, stromal cells, and osteoblasts (Figure 1F).12Holmbeck K Bianco P Pidoux I Inoue S Billinghurst RC Wu W Chrysovergis K Yamada S Birkedal-Hansen H Poole AR The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone.J Cell Sci. 2005; 118: 147-156Crossref PubMed Scopus (173) Google Scholar, 22Filanti C Dickson GR Di Martino D Ulivi V Sanguineti C Romano P Palermo C Manduca P The expression of metalloproteinase-2, -9, and -14 and of tissue inhibitors-1 and -2 is developmentally modulated during osteogenesis in vitro, the mature osteoblastic phenotype expressing metalloproteinase-14.J Bone Miner Res. 2000; 15: 2154-2168Crossref PubMed Scopus (68) Google Scholar, 23Sato T del Carmen Ovejero M Hou P Heegaard AM Kumegawa M Foged NT Delaisse JM Identification of the membrane-type matrix metalloproteinase MT1-MMP in osteoclasts.J Cell Sci. 1997; 110: 589-596Crossref PubMed Google Scholar, 24Takagi M Santavirta S Ida H Ishii M Akimoto K Saotome K Konttinen YT The membrane-type-matrix metalloproteinase/matrix metalloproteinase-2/tissue inhibitor of metalloproteinase-2 system in periprosthetic connective-tissue remodeling in loose total-hip prostheses.Lab Invest. 1998; 78: 735-742PubMed Google Scholar LNCaP cells, which lack endogenous MT1-MMP,25Nagakawa O Murakami K Yamaura T Fujiuchi Y Murata J Fuse H Saiki I Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) on prostate cancer cell lines.Cancer Lett. 2000; 155: 173-179Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar were stably transfected with a vector expressing full-length wild-type human MT1-MMP. As a control, LNCaP cells were also transfected with an empty vector. Expression of MT1-MMP was confirmed by reverse transcriptase-PCR (RT-PCR) and immunoblotting (Figure 2, A and B, respectively) in the LNCaP cells transfected with MT1-MMP cDNA (LNCaP-MT1wt) but not in the control cells (LNCaP-Neo). In accordance with studies from other groups,25Nagakawa O Murakami K Yamaura T Fujiuchi Y Murata J Fuse H Saiki I Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) on prostate cancer cell lines.Cancer Lett. 2000; 155: 173-179Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26Zhang J Jung K Lein M Kristiansen G Rudolph B Hauptmann S Schnorr D Loening SA Lichtinghagen R Differential expression of matrix metalloproteinases and their tissue inhibitors in human primary cultured prostatic cells and malignant prostate cell lines.Prostate. 2002; 50: 38-45Crossref PubMed Scopus (29) Google Scholar MT1-MMP mRNA and protein were nondetectable in parental LNCaP cells. Whole-cell lysates of LNCaP-MT1wt cells contained 60- and 57-kd proteins representing the latent and active forms of MT1-MMP, respectively.18Hernandez-Barrantes S Toth M Bernardo MM Yurkova M Gervasi DC Raz Y Sang QA Fridman R Binding of active (57 kDa) membrane type 1-matrix metalloproteinase (MT1-MMP) to tissue inhibitor of metalloproteinase (TIMP)-2 regulates MT1-MMP processing and pro-MMP-2 activation.J Biol Chem. 2000; 275: 12080-12089Crossref PubMed Scopus (272) Google Scholar An antibody-based enzymatic activity assay specific for MT1-MMP demonstrated that LNCaP-MT1wt cells exhibited significantly higher activity than LNCaP-Neo cells (Figure 2C). A series of in vitro assays were done to confirm the functionality of the ectopically expressed MT1-MMP. First, we tested the capacity of LNCaP transfectant cells to activate exogenous pro-MMP-2, because these cells did not express endogenous MMP-2.26Zhang J Jung K Lein M Kristiansen G Rudolph B Hauptmann S Schnorr D Loening SA Lichtinghagen R Differential expression of matrix metalloproteinases and their tissue inhibitors in human primary cultured prostatic cells and malignant prostate cell lines.Prostate. 2002; 50: 38-45Crossref PubMed Scopus (29) Google Scholar As expected, only LNCaP-MT1wt cells were capable of fully activating exogenous reco
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