Oncogene (2008) 27, 6347–6355 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Matrix -11/stromelysin-3 exhibits collagenolytic function against collagen VI under normal and malignant conditions

ER Motrescu1, S Blaise1, N Etique1, N Messaddeq1, M-P Chenard2, I Stoll1, C Tomasetto1 and M-C Rio1

1Departement de Biologie du Cancer, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS UMR 7104, INSERM U596, Universite´ Louis Pasteur, Illkirch Cedex, France and 2Service d’Anatomie Pathologique Ge´ne´rale, Centre Hospitalier Universitaire de Hautepierre, Strasbourg Cedex, France

The substrate of 11 (MMP11) remodeling during embryogenesis, wound healing, tissue remains unknown. We have recently shown that MMP11 involution and metamorphosis (Rio, 2002). More is a negative regulator of adipogenesis, able to reduce and importantly, MMP11 is expressed by the peritumoral even to revert mature adipocyte differentiation. Here, we fibroblasts, and high levels of expression correlate with have used mouse 3T3L1 cells and human U87MG and tumor aggressiveness as well as with poor patient clinical SaOS cells to show that MMP11 cleaves the native a3 outcome (Basset et al., 1990; Basset et al., 1997). In vivo chain of collagen VI, which is an adipocyte-related studies using different mouse tumor models showed that component. It is known that extra- MMP11 plays a key role in tumor development (Masson cellular proteolytic processingof this chain is required et al., 1998; Noel et al., 2000) and that it acts at the for correct collagen VI folding. Interestingly, MMP11- initial step of the invasive processes (Boulay et al., 2001). deficient fat tissue is less cohesive and exhibits collagen VI At this step, the invasive cancer cells establish an alteration, dramatic adipocyte plasma and basement epithelial/mesenchymal heterotypic cell interaction in- membrane abnormalities and lipid leakage. MMP11 is ducing dramatic remodeling of the adjacent connective thus required for correct collagen VI folding and therefore tissue through desmoplasia, leading to the formation of for fat tissue cohesion and adipocyte function. Both the stroma. One of the major changes seen during MMP11 and collagen VI favor tumor progression. desmoplasia is the accumulation of fibroblasts and the Similar spatio-temporal overexpression at the adipo- disappearance of adipocytes. Recently, the relevance of cyte–cancer cell interface has been reported for the two adipocytes and adipocyte-derived factors to cancer cell proteins. MMP11-dependent collagen VI processing survival and growth has started to receive increasing might therefore be expected to occur during malignancy. attention (Manabe et al., 2003; Celis et al., 2005; Accordingly, collagen VI no longer delineates adipocytes Dieudonne et al., 2006; Schaffler et al., 2007). We located at the invasive front of breast carcinomas. In recently showed that MMP11 is a negative regulator of conclusion, the native a3 chain of collagen VI constitutes a adipogenesis not only able to reduce preadipocyte specific MMP11 substrate. This MMP11 collagenolytic differentiation but also to revert mature adipocytes into activity is functional in fat tissue ontogenesis as well as preadipocytes (Andarawewa et al., 2005). This function duringcancer invasive steps. is aberrantly restored in carcinomas. Thus, invasive Oncogene (2008) 27, 6347–6355; doi:10.1038/onc.2008.218; cancer cells at the tumor invasive front induce proximal published online 14 July 2008 adipocytes to express MMP11 inducing therefore the dedifferentiation of peritumoral adipocytes into fibro- Keywords: matrix metalloproteinase-11/stromelysin-3; blasts. Thus, at an early stage of tumor invasion, collagen VI; adipocyte; fat tissue; breast cancer MMP11 helps the implantation of cancer cells into adjacent connective tissue via cancer cell–adipocyte interaction (reviewed in Motrescu and Rio, 2008). Introduction Despite extensive research from several laboratories using various approaches, the specific substrate of Matrix metalloproteinase-11 (MMP11)/stromelysin-3 is MMP11 has not yet been found. In this context, a zinc endopeptidase belonging to the MMP family. adipocytes might offer the answer to this question. This has been shown to be involved in tissue One good candidate which could be a target of MMP11 is collagen VI that has been shown to be expressed and Correspondence: Dr M-C Rio, Departement de Biologie du Cancer, secreted abundantly by adipocytes, and to be involved in Institut de Ge´ ne´ tique et de Biologie Mole´ culaire et Cellulaire, CNRS cancer cell–adipocyte heterotypic signaling (Iyengar UMR 7104, INSERM U596, Universite´ Louis Pasteur, BP 10142, et al., 2003; Iyengar et al., 2005). Collagen VI has a Illkirch Cedex 67404, France. E-mail: [email protected] complex structure containing three different chains, a1 Received 30 April 2008; revised 3 June 2008; accepted 16 June 2008; (around 150 kDa), a2 (around 150 kDa) and a3 (around published online 14 July 2008 250 kDa) that associate intracellularly in triple helical Collagen VI is a substrate of MMP11 ER Motrescu et al 6348 monomers, dimers and tetramers. Once secreted, the tetramers associate end-to-end into microfibrils (Aigner et al., 2002). Collagen VI participates in the local extracellular matrix (ECM) environment by providing structural support for cells and enrichment of various molecules, and can itself assume important signaling effects (Ruhl et al., 1999). Thus, it has been reported that adipocytes play a vital role in defining the ECM environment for normal and tumor-derived ductal epithelial cells and contribute significantly to tumor growth at early stages through secretion and processing of collagen VI (Iyengar et al., 2003; Iyengar et al., 2005). In the present study, we have investigated this hypothesis both in vitro and in vivo. We demonstrate that MMP11 indeed cleaves collagen VI. Only the native a3 chain of collagen VI is degraded by MMP11. Moreover, the absence of MMP11 in deficient mice Figure 1 Silver nitrate staining and western blot analysis of matrix dramatically alters the collagen VI folding and subse- metalloproteinase 11 (MMP11) effect on pepsinized collagen VI. (a) Silver nitrate staining of a1 proteinase inhibitor (a1-PI) alone quently adipocytes and related ECM. Finally, the (lane 1) or incubated for 4.5 h with active (lane 2) or inactive (lane ectopic expression of MMP11 at the adipocyte–cancer 3) mouse recombinant MMP11 proteins. Active MMP11 cleaves cell interface leads to collagen VI alteration. the a1-PI, but not the inactive form. (b) Silver nitrate staining of purified collagen VI pepsinized protein alone (lane 1) or incubated for 4.5 h with active (lane 2) or inactive (lane 3) mouse recombinant MMP11 (c) using LB-1697 antibody directed against collagen VI, Results western blot analysis of purified collagen VI pepsinized protein alone (lane 1) or incubated for 4.5 h with active (lane 2) or inactive (lane 3) mouse recombinant MMP11. MMP11 has no effect on MMP11 does not cleave purified pepsin-solubilized soluble collagen VI. Molecular sizes of collagen VI fragments are collagen VI indicated in kDa. The capacity of MMP11 to cleave collagen VI was first investigated in test-tube experiments using recombinant MMP11 and commercially available purified human only a low level of collagen VI (Figure 2A upper part, pepsin-solubilized collagen VI protein (Abcam, UK). lane 1), abundant collagen VI levels were shown at day The enzymatic efficiency of recombinant MMP11 10 adipocyte-differentiated 3T3L1 (Figure 2A upper protein was proved by using the a1 proteinase inhibitor part, lane 2). We observed two main bands at around (a1-PI) that has been shown to be degraded by MMP11 250 and 150 kDa that presumably correspond to the a3 (Pei et al., 1994). Silver nitrate staining showed that the chain and to the a1 and a2 chains, respectively (Aigner a1-PI band disappeared in the presence of active MMP11 et al., 2002). The differentiation of the 3T3L1 fibroblasts (Figure 1a). Pepsin-solubilized collagen VI gave rise to (Figure 2Ba) into adipocytes (Figure 2Bb) was visua- three main bands at around 40, 50 and 60 kDa as shown lized by Oil Red O staining of lipid droplets. Moreover, by silver nitrate staining (Figure 1b) and by western blot using the 5ST-4A9 monoclonal antibody directed using the rabbit anti-collagen VI polyclonal antibody against MMP11, western blot analysis revealed only a LB-1697 (Gentaur, France; Figure 1c). Addition of mouse very low level of endogenous MMP11 in confluent active (lane 2) or inactive (lane 3) recombinant MMP11 3T3L1 fibroblasts, but not in adipocyte-differentiated did not affect the purified collagen VI protein (Figures 1b 3T3L1 (Figure 2A, lower part, lanes 1 and 2, respec- and c). MMP11 has thus no enzymatic function against tively). Thus, 3T3L1 differentiation into adipocyte is pepsin-generated collagen VI fragments. associated with an increase of collagen VI and a decrease of MMP11. These data suggested that MMP11 might have a function on native collagen VI. MMP11 acts on native collagen VI produced by DM-induced adipocyte-differentiated mouse 3T3L1 cells As there was no effect of MMP11 on pepsin-digested MMP11 treatment of adipocyte-differentiated 3T3L1 collagen VI, we were prompted to find some sources of leads to their dedifferentiation and to a decrease of native collagen VI to avoid the pepsination step that is collagen VI involved in the purification of commercialized collagen As no endogenous MMP11 was expressed in adipocyte- VI. We therefore induced adipocyte differentiation in differentiated 3T3L1, we took advantage of this model canonical mouse 3T3L1 fibroblasts that become pre- to test the impact of MMP11 on native collagen VI. At adipocytes when they reach confluence. Treatment of day 10 after the start of differentiation, active or inactive confluent cells with a differentiation-inducing mix (DM) forms of recombinant mouse MMP11 (3.5 mg/ml) were induces the expression of proteins associated with added over 72 h to the culture medium of adipocyte- mature adipocytes and the accumulation of lipids differentiated 3T3L1. Subsequently, the cells were (Bernlohr et al., 1984). Whereas the concentrated culture cultured for 24 h with the medium without fetal calf medium from confluent 3T3L1 fibroblasts expressed serum (FCS). The recombinant active MMP11 induces a

Oncogene Collagen VI is a substrate of MMP11 ER Motrescu et al 6349 cell lines using recombinant MMP11. The human cells used were the glioblastoma U87MG cell line that has been shown to produce intact collagen VI including the a1 and a2 chains around 150 kDa each and the a3 chain around 250 kDa (Han and Daniel, 1995), and the SaOS2 cell line that is deficient in production of a3 chain (Lamande et al., 2006). Cell-conditioned culture media were collected and concentrated (see ‘Materials and methods’). Western blot analysis showed two bands at around 150 and 250 kDa in U87MG and only one band at around 150 kDa for SaOS2 as was expected (Figure 4, lane 1). The active and inactive forms of recombinant MMP11 were visualized using western blot and 5ST-4C10 monoclonal antibody (Figure 4, lanes 2 and 3, respec- tively). Note that autolysis is observed for the MMP11 active form (two bands) but not for the inactive one (one band). The concentrated cell-conditioned culture media were incubated with recombinant active or inactive MMP11 for 4.5 h at 37 1C at a ratio 5:1 w/w. In the presence of active MMP11, a degradation of the 250 kDa band was observed in U87MG. The 150 kDa band from the two cell lines however was unaffected (Figure 4, lane 2). No effect was observed in the case of incubation of culture media with inactive MMP11 (Figure 4, lane 3). Thus, MMP11 does not affect the a1anda2 chains of collagen VI, but specifically cleaves the a3chain.

Figure 2 Western blot analysis of endogenous matrix metallo- proteinase 11 (MMP11) and collagen VI produced by day 10 Mouse MMP11-deficient fat tissue shows collagen VI adipocyte-differentiated 3T3L1. (A) Western blot analysis using alteration concentrated culture medium from 3T3L1 cells at confluence (lane To investigate the proteolytic function of MMP11 on 1) or at day 10 after differentiation-inducing mix (DM) start (lane collagen VI in vivo, we first compared the collagen 2). Collagen VI is shown using LB-1697 antibody in the upper panel, and endogenous MMP11 expression using the 5ST-4A9 VI present in adipose tissue of wild-type and antibody in the lower panel. Molecular sizes of endogenous MMP11-deficient mice previously developed in the collagen VI chains and MMP11 are indicated in kDa. (B) laboratory (Masson et al., 1998). Immunohisto- Adipocyte differentiation was visualized using Oil Red O staining chemistry was performed on abdominal fat from of 3T3L1 cells at confluence (panel a) and at day 10 after DM start 8-week-old wild-type or MMP11-deficient mice using (panel b). Collagen VI is highly expressed and secreted by adipocyte-differentiated 3T3L1 cells, whereas no endogenous the LB-1697 anti-collagen VI polyclonal antibody. MMP11 is detectable. Collagen VI was expressed in both animals, but the staining gave different images. In wild-type fat tissue, collagen VI staining appeared as a thin compact decrease in lipid droplets as shown by Oil Red O continuous band surrounding adipocytes (Figure 5a). staining (Figure 3Bb), indicating that it induces the In MMP11-deficient mice, two types of collagen VI dedifferentiation of 3T3L1 adipocytes as already shown staining were observed. First, an enlarged and diffuse using a primary culture of mouse embryonic fibroblasts band surrounding adipocytes was seen (Figure 5b), (Andarawewa et al., 2005). The expression of collagen indicating a fault in collagen VI folding. Second, this VI was checked in the concentrated culture media using staining was discontinuous and sometimes even totally western blot analysis (Figure 3A). A decrease in the lost at the cell–cell contact between two adjacent collagen VI level was observed in the presence of active adipocytes, indicating that collagen VI fibrils were recombinant MMP11 (Figure 3A, lane 2). Neither disrupted. Strong staining however remained present reduction of lipid droplets nor the collagen VI level at the triangular interspaces existing between several was observed using the inactive recombinant MMP11 adjacent adipocytes (Figure 5c). Finally, it might be (Figures 3A and Bc, lane 3), similar to the control expected that misfolded and unstable collagen VI could (Figures 3A and Ba, lane 1). These data strongly be unfunctional leading to ECM alteration. Accord- suggested that MMP11 cleaves the native collagen VI. ingly, optical microscopic examination showed that adipose tissues were less cohesive in MMP11-deficient (Figures 5b and c) than in wild-type (Figure 5a) mice. MMP11 cleaves the a3 chain of native collagen VI Overall these results show that the absence of MMP11 To confirm this hypothesis, we performed test-tube leads to alteration in fat tissue collagen VI folding and digestion of native collagen VI produced by two human stability.

Oncogene Collagen VI is a substrate of MMP11 ER Motrescu et al 6350

Figure 3 Western blot analysis of the impact of matrix metalloproteinase 11 (MMP11) treatment on day 10 adipocyte-differentiated 3T3L1. (A) Western blot analysis of collagen VI expression using LB-1697 antibody in the concentrated culture medium from 3T3L1 adipocytes at day 10 after differentiation-inducing mix (DM) start either alone (lane 1) or treated for 72 h with 3.5 mg/ml mouse recombinant MMP11 protein active (lane 2) or inactive (lane 3). Molecular sizes of endogenous collagen VI chains are indicated in kDa. Red ponceau staining serves as loading control. (B) Oil Red O staining of lipid droplets in 3T3L1 adipocytes at day 10 after differentiation start (panel a) or incubated for 72 h with mouse recombinant MMP11 active (panel b) or inactive (panel c). Active MMP11 treatment leads to a decrease of collagen VI associated with the 3T3L1 dedifferentiation. Inactive MMP11 has no effect.

Electron microscopy highlights numerous ultrastructural alteration of mouse MMP11-deficient adipocytes and related ECM We then performed electron microscopy to further visualize modifications occurring in the fat tissue of MMP11-deficient mice. The ultrastructural analysis showed that the ECM present at the interface between adipocytes was greatly altered. Whereas correctly folded continuous parallel collagen fibers were observed in wild-type mice (Figures 6a and b), MMP11- deficient fat tissue showed disorganized and disrupted Figure 4 Western blot analysis of the impact of matrix collagen structures (Figures 6c and d). In addition, metalloproteinase 11 (MMP11) on native collagen VI collected empty areas were observed. Moreover, alterations of from U87MG and SaOS2 human cell lines. Western blot analysis adipocyte ultrastructure were also observed, which (LB-1697 antibody) of collagen VI present in concentrated culture included disruption of adipocyte basement membrane, media conditioned using U87MG or SaOS2 cells as indicated on the left, either alone (lane 1) or after having been incubated for disruption and extension of adipocyte plasma mem- 4.5 h at 37 1C with active (lane 2) or inactive (lane 3) recombinant brane (Figures 6g and h). Lipid leakage leading to MMP11 protein. For incubation the ratio 5:1 (w/w, concentrated lipid accumulation outside adipocytes and notably culture medium to recombinant MMP11) was used. Recombinant infiltration of lipid droplets into the cytoplasm of active (lane 2) and inactive (lane 3) MMP11 proteins were visualized using the 5ST-4C10 monoclonal antibody. Note that adjacent cells and in ECM were also present (Figures two bands were observed for the MMP11 active form due to its 6f and g). None of these modifications were observed in autolysis. Active MMP11 treatment has no effect on the 150 kDa the fat tissue from wild-type mice (Figure 6e). Incorrect band of the two cell lines. The intensity of the staining of the collagen VI structure cause by the absence of MMP11 250 kDa band of U87MG is however dramatically decreased, thus appears to lead to the alteration of both plasma indicating that it is cleaved by MMP11. Inactive MMP11 has no effect. The molecular sizes of endogenous collagen VI chains and and basement membranes of adipocytes and adipocyte- recombinant MMP11 are indicated in kDa. related ECM.

Oncogene Collagen VI is a substrate of MMP11 ER Motrescu et al 6351

Figure 5 Immunohistochemical analysis of collagen VI present in fat tissue from wild-type and matrix metalloproteinase 11 (MMP11)-deficient mice. Collagen VI immunohistochemical analysis (LB-1697 antibody) of sections of adipose tissue from wild-type (a) and MMP11-deficient mice (b, c). Abdominal fat tissue was obtained from 8-week-old mice. Whereas collagen VI staining of wild- type fat tissue gives rise to a thin compact continuous line, an enlarged band either continuous or disrupted is seen around MMP11- deficient adipocytes. (a–c) Magnification: Â 1000.

Figure 7 Immunohistochemical analysis of collagen VI and matrix metalloproteinase 11 (MMP11) at the tumor invasive front of human breast cancer. Collagen VI immunohistochemistry (LB-1697 antibody) of normal breast adipose tissue (a) and invasive front of breast tumor (b). MMP11 immunohistochemistry (5ST-4A9 antibody) of normal breast adipose tissue (c) and the invasive front of breast tumor (d). Normal fat tissue located further away from the tumor is collagen VI positive and MMP11 negative. As previously described, adipocytes located at the tumor invasive front exhibit strong MMP11 expression and a considerable reduction of their sizes (Andarawewa et al., 2005). They were no longer surrounded by the thin compact line of collagen VI observed in resting adipocytes (a). A more or less homogenous mild or very Figure 6 Electron microscopy analysis of fat tissue from wild-type low collagen VI staining is observed in the extracellular matrix and matrix metalloproteinase 11 (MMP11)-deficient mice. Electron (ECM, b). (a–d) Magnification: Â 400. microscopy analysis shows that the collagen structures present at the interface between adipocytes are altered in MMP11-deficient fat tissue (c, d) compared with wild type (a, b). Moreover, in MMP11-deficient mice, disruption of adipocyte basement mem- invasive front of human breast carcinoma are induced brane, disruption and extensions of the plasma membrane, and by invasive cancer cells to strongly express MMP11, and lipid droplets infiltration in adjacent extracellular matrix (ECM) exhibit dramatically reduced sizes. It was therefore and cells are observed (f–h). In the wild-type mice, none of these modifications are observed (e). Magnification: bar 1 mm(a, c, e, f), tempting to speculate that, at the tumor invasive front, 0.5 mm(b, d). the ectopically expressed and secreted MMP11 might alter collagen VI normally present around adipocytes. We therefore checked the patterns of collagen VI and MMP11 participates in collagen VI alteration MMP11 on adjacent sections of human breast tumors at the human tumor invasive front using immunohistochemistry. As expected, we observed All the results collected above prompted us to investi- that the adipocytes located at the invasive front gate the possible involvement of this collagenolytic abnormally expressed high levels of MMP11 (Figures function of MMP11 in the invasive phase of tumor 7d). Interestingly, they were no longer surrounded by progression. Normal resting human adipocytes ex- the thin compact line of collagen VI (Figure 7b) pressed collagen VI (Figure 7a) but not MMP11 normally observed in resting adipocytes (Figure 7a). A (Figure 7c). However, as shown in our previous study more or less homogenous mild or very low collagen VI (Andarawewa et al., 2005), adipocytes located at the staining (Figures 7b) was observed in the ECM. These

Oncogene Collagen VI is a substrate of MMP11 ER Motrescu et al 6352 data indicate that MMP11 alters collagen VI at the commercially available pepsin-solubilized collagen VI. tumor invasive front. MMP11 however cleaves the native a3 chain of collagen VI produced by the human glioblastoma cell line U87MG, but not the a1anda2 chains. Accordingly, Discussion no MMP11 effect was observed on collagen VI produced by the osteosarcoma cell line SaOS-2, known An increasing number of studies highlight the impor- to be devoid of a3 chain expression (Lamande et al., tance of interactions between cancer cells and adipocytes 2006). The precise site of MMP11 cleavage remains to in tumor development (Manabe et al., 2003; Celis et al., be determined. Collectively, these data clearly indicate 2005; Dieudonne et al., 2006; Schaffler et al., 2007). We that MMP11 possesses a collagenolytic function against have recently shown that MMP11 is involved in these the native a3 chain of collagen VI. processes (Andarawewa et al., 2005). Investigations MMP11 belongs to the MMP family whose members performed by Scherrer’s group in the field focused on share the ability to cleave one or several matrix the role played by collagen VI in tumor progression components. Collagens constitute a very large family (Iyengar et al., 2003; Iyengar et al., 2005). We provide a including more than 20 members. Among them, the link between these two molecules in the present study main collagen types reported to be cleaved by MMP and show that MMP11 possesses collagenolytic function members are the types I and II (reviewed in (McCawley against the native a3 chain of collagen VI under both and Matrisian, 2001)). At the moment, very few data are normal and malignant conditions. available concerning collagen VI and MMPs. It has been Despite numerous studies, no matrix substrate has reported that MMP1, MMP2, MMP3 and MMP9 are been identified for the MMP11. Recently, we have unable to cleave intact collagen VI microfibrils (Kielty shown that MMP11 is a potent negative regulator of et al., 1993). MMP2 however cleaves the a3 chain of the adipogenesis, able to reduce and even to revert human corneal collagen VI whose disulfide bonds have adipocyte differentiation in an autocrine manner (An- been reduced (Myint et al., 1996). Thus, collagen VI is darawewa et al., 2005). It was therefore tempting to not a common substrate for MMPs. This substrate speculate that the MMP11 substrate might belong specificity furthermore highlights the originality of to adipocyte-related ECM. In this context, collagen VI MMP11 among the other MMPs (Rio, 2002). is a protein highly enriched in adipocytes (Iyengar et al., It has been reported that the a3 chain is essential for 2003). Thus, one of the important changes in the the formation of stable collagen VI. The C-terminal C5 transition from fibroblast-like phenotype to adipocytes domain of the a3 chain is critical for the interactions in culture models is the increase in collagen VI between tetramers that promote efficient microfibril (Nakajima et al., 2002). Accordingly, we observed an formation (Lamande et al., 2006). Thus, in articular increase in collagen VI secreted by the mouse 3T3L1 cartilage and presumably also in other tissues a simultaneously to their DM-induced adipocyte differ- maturation process of collagen VI fibrils takes place to entiation. If the acquirement of adipocyte phenotype a significant extent in the immediate pericellular matrix induces an increase in the level of collagen VI, then we compartment through proteolytic processing of the C5 would expect that the MMP11-dependent dedifferentia- domain of the a3 chain that is not present in the mature tion of adipocytes will be associated with a decrease of pericellular collagen VI (Aigner et al., 2002). The nature collagen VI levels. As expected, we showed that the of the proteinase involved remains unknown. From our reduction in the number and size of lipid droplets, which data, we propose that MMP11 plays this role. This occurs when the adipocyte-differentiated 3T3L1 are hypothesis is supported by the immunohistochemical incubated with recombinant active MMP11, correlates analysis of fat tissue from MMP11-deficient mice, with the decrease in the collagen VI present in the showing enlarged uncorrectly folded collagen VI sur- culture medium. These data indicated that there is a rounding adipocytes or disruption of collagen VI at the relationship between MMP11 and collagen VI. adipocyte–adipocyte interface. MMP11 is thus required We therefore investigated the possible cleavage of in vivo for correct collagen VI folding. collagen VI by MMP11. Collagen VI is composed of It might be hypothesized that incorrectly folded and three genetically distinct a-chain subunits, a1, a2and fragile collagen VI has lost, at least partially, its a3, each of which contains a relatively short triple helix, functionality. Accordingly, the fat tissue was less and N- and C-terminal globular regions. The a3 chain is cohesive in MMP11-deficient mice than in wild-type much larger than the other two (250 versus 150 kDa). mice. Electron microscopy ultrastructural analysis The precise protein interactions initiating and regulating showed alteration of collagen network associated with formation of the unique collagen VI microfibril supra- disruption of adipocyte basement and plasma mem- molecular assemblies are not known, but there are branes. Interestingly, Kuo et al. (1997) showed that several steps. Following heterotrimeric assembly of the collagen VI is involved in cell adhesion by a direct a1, a2 and a3, these monomers form higher order interaction with collagen IV, one important component structures intracellularly by aligning in an antiparallel of the basement membranes. Finally, MMP11-deficient manner first to form dimers and then tetramers by adipocytes become unable to retain lipids, allowing lipid lateral association of dimers. After secretion, tetramers accumulation in aberrant extracellular areas and adja- associate end-to-end to form microfibrils (Aigner et al., cent cells. Thus, the absence of MMP11 finally leads to 2002). We showed that MMP11 does not cleave the alteration of the functionality of adipose tissue. As

Oncogene Collagen VI is a substrate of MMP11 ER Motrescu et al 6353 mature adipocytes are generally considered incapable or xanthine) was added. After 48h, the DM was replaced with limited of division (Gregoire, 2001), our data indicate fresh culture medium containing 10 mg/ml insulin. Cell that MMP11 is involved in fat tissue ontogenesis. differentiation was followed by Oil Red O staining. Another important function of the collagen VI has been reported in cancers. Breast cancer is associated Oil red O staining with an increase in collagen VI expression at the level of The cells were fixed with 10% formalin in phosphate-buffered the peritumoral adipocytes. Moreover, collagen VI- saline (PBS) 1 Â . After 10 min, the cells were washed three times with distilled water, air-dried and incubated at room deficient mice show reduced rates of MMTV-PyMT- temperature (RT) with 0.5% Oil Red O for 20 min. The cells induced mammary tumors (Iyengar et al., 2005). were then washed again with distilled water and counterstained Interestingly, it has been shown that the a3 chain of with hematoxylin as described previously (Andarawewa et al., collagen VI is cleaved within breast tumors. Once again, 2005). MMP11 might be expected to be involved. Indeed, under normal condition, resting adipocytes express Expression and purification of mouse recombinant MMP11 collagen VI and are surrounded by basement membrane proteins mainly composed of collagen VI, but they express very Mouse recombinant active (F102-S276) and inactive MMP11 little MMP11. We have however previously demon- (same sequence with Glu220Ala mutation) were constructed in strated that invasive cancer cells induce strong MMP11 the pET3b vector. The plasmids were transformed into the expression by adjacent adipocytes (Andarawewa et al., expressing Escherichia coli strain BL21(DES)pLysS. The 2005). Thus, invasive processes aberrantly induce mutant proteins were obtained from insoluble protein fraction of E. coli using the previously described protocol (Kannan simultaneous spatio-temporal overexpression and secre- et al., 1999). Briefly, after production, the bacterial cells were tion of collagen VI and MMP11. In contrast to most of collected and treated to obtain bacterial inclusion bodies. the other MMPs that need to be activated extracellu- Proteins were recovered, refolded and purified from inclusion larly, MMP11 is secreted in an enzymatically active bodies. Characteristics of the proteins thereby produced have form (Pei and Weiss, 1995), MMP11 collagenolytic been previously described (Noel et al., 1995). function can therefore rapidly occur at the interface The enzymatic activity of active and inactive MMP11 was between adipocytes and invasive cancer cells. Accord- assessed using the quantitative colorimetric substrate assay ingly, we observed the disappearance of the thin with a1-PI (Kannan et al., 1999). Briefly, the MMP11 and compact collagen VI lining of adipocytes located at a1-PI incubation was left for 1 h at RT and this mixture the invasive front of human breast cancer. Both collagen was subsequently incubated with a-chymotrypsin for 20 min. Enzymatic activity was visualized by adding N-succinyl-Ala- VI and MMP11 have been shown to favor tumor Ala-Pro-Phe-p-nitroanilide synthetic substrate for 2 min. The progression. In this context, MMP11-dependent clea- optical density at 405 nm was then determined (Beckman vage of collagen VI might generate an increase of a DU640 spectrometer; Fullerton, CA, USA). C-terminal a3 chain proteolytic fragment that has been shown to possess potent growth stimulatory effects on Dedifferentiation of 3T3L1 adipocytes cancer cells (Iyengar et al., 2005). Moreover, we have At day 10 after the start of differentiation, active or inactive previously shown that cancer cell-induced MMP11 mouse recombinant MMP11 protein was added to the culture expression by adjacent adipocytes leads to their ded- medium for 72 h at a concentration of 3.5 mg/ml. The medium ifferentiation and the accumulation of peritumoral was changed every 24 h as recombinant MMP11 is susceptible fibroblast-like cells, which are well known to promote to autolysis (Noel et al., 1995). tumor progression (Motrescu and Rio, 2008). Collec- tively, the present findings suggest that the event Immunohistochemistry initiating this adipocyte dedifferentiation might be the Sections of human breast tumor invasive front and adjacent cleavage of collagen VI by MMP11. normal fat tissue were immunostained using the 5ST-4A9 mouse monoclonal antibody directed against MMP11 (IGBMC; Euromedex, Illkirch, France) as described pre- viously (Basset et al., 1990) and the LB-1697 anti-collagen VI Materials and methods rabbit polyclonal antibody (Gentaur) directed against collagen VI extracted from placenta. LB-1697 recognizes the human Cell lines and mouse a1, a2 and a3 collagen VI chains; however, the Mouse fibroblast 3T3L1, human glioblastoma U87MG and recognized epitopes remain unknown. human osteosarcoma SaOS-2 cell lines were obtained from To perform immunohistochemistry on mouse abdominal fat American Type Culture Collection (Molsheim, France). The tissue, adipose tissue from five wild-type and five MMP11- 3T3L1 and SaOS-2 cell lines were cultured in Dulbecco’s modified deficient mice was collected, embedded in paraffin and Eagle’s medium (Gibco, Invitrogen Corporation, UK), 10% FCS analysed using the LB-1697 polyclonal rabbit antibody for and gentamicin. The U87MG cell line was cultured in minimum collagen VI (Gentaur). essential medium (Gibco, Invitrogen Corporation, UK) with 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10% FCS and SDS–PAGE and western blot gentamicin. The cells at confluence were incubated for 24 h using medium without FCS. After 24 h of incubation, the cell culture medium Differentiation of 3T3L1 cells into adipocytes was collected, centrifuged at 160 g for 5 min to remove cell The 3T3L1 cells were grown until they reached confluence. debris, and kept frozen at minus 80 1C until use. Before Subsequently, a differentiation inducing mix (DM, 10 mg/ml analysis, the culture medium was thawed and subsequently insulin, 0.5 mmol/l dexamethasone, 0.5 mmol/l methylisobutyl- 0.1% of the detergent ((3-cholamidopropyl)dimethylammo-

Oncogene Collagen VI is a substrate of MMP11 ER Motrescu et al 6354 nio)-1-propane sulfonate) was added. Afterward the culture 10 min in darkness was necessary to stain the gel using medium was concentrated by a factor of 15 using Centricon a 0.1% solution of silver nitrate. The gel was washed 10 kDa separation membranes (Millipore Corporation, with water and developed in the presence of a solution Bedford, MA, USA) by centrifugation at 2500 g. The protein of 2% potassium bicarbonate, 0.008% formaldehyde. The concentration of each sample was calculated using a protein reaction was stopped using a 10% ethanol and 5% acetic assay kit (Bio-Rad, France) and a Beckman Spectophotometer acid solution. (Beckman Instruments, France). The concentrated sample was incubated with recombinant MMP11 at a ratio of 5:1 (w/w; Electron microscopy total protein of culture medium/recombinant MMP11) for For electron microscopy analysis, the tissue samples were fixed 4.5 h at 37 1C. Subsequently, the samples were prepared with in 2.5 glutaraldehyde in cacodylate buffer overnight at 4 1C, SDS sample buffer and 2-mercaptoethanol, boiled for 7 min washed for 30 min in the same buffer and then postfixed for 1 h and fractionated by SDS–polyacrylamide gel electrophoresis. in 1% buffered osmium tetroxide. The samples were dehy- The gels were electrotransferred to nitrocellulose membranes drated with gradient concentrations of ethanol and embedded (Schleicher & Schuell, Dassel, Germany), blocked with PBS in Epon 812. Ultrathin sections were cut, stained with uranyl containing 3% nonfat dry milk and 0.1% Tween 20 and acetate and lead citrate according to standard procedures for incubated with specific primary antibodies (collagen VI, LB- electron microscopy. A Morgagni 268D type microscope was 1697; endogenous MMP11, 5ST-4A9; recombinant MMP11, used for the analysis. 5ST-4C10). Horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as secondary antibodies. Immunoreactive bands were Acknowledgements visualized using an enhanced chemiluminescence detection system (Amersham Biosciences, Arlington Heights, IL, USA) We thank Takako Sasaki from the Max-Planck-Institute for and Kodak BioMax MR Film (Sigma-Aldrich, St Louis, Biochemistry, Munich, Germany for his help. This study was MO, USA). supported by funds from the Institut National de la Sante´ et de la Recherche Me´ dicale, the Centre National de la Recherche Silver nitrate staining Scientifique, the Hoˆ pital Universitaire de Strasbourg, the After the migration of the proteins on SDS-polyacrylamide Association pour la Recherche sur le Cancer, the Ligue gel, the gel was incubated overnight in a solution of 50% Nationale Franc¸aise contre le Cancer and the Comite´ sdu ethanol, 10% acetic acid followed by 10 min incubation with a Haut-Rhin et du Bas-Rhin (e´ quipe labellise´ e), the European solution of 50% ethanol, 5% acetic acid and 2% copper Commission (FP6 LSHC-CT-2003-503297; Cancer Degra- chloride. Afterward the gel was incubated with 0.01% dome Project), the Institut National du Cancer, Fond National potassium permanganate solution, washed with 10% ethanol pour la Sante´ ACI 2004 Cance´ ropoˆ le Grand-Est and the solution and then with water. An incubation time period of ‘Ruban Rose’ prize. EM was a recipient of an EU fellowship.

References

Aigner T, Hambach L, Soder S, Schlotzer-Schrehardt U, Poschl E. Dieudonne MN, Bussiere M, Dos Santos E, Leneveu MC, Giudicelli (2002). The C5 domain of Col6A3 is cleaved off from the Col6 fibrils Y, Pecquery R. (2006). Adiponectin mediates antiproliferative and immediately after secretion. Biochem Biophys Res Commun 290: apoptotic responses in human MCF7 breast cancer cells. Biochem 743–748. Biophys Res Commun 345: 271–279. Andarawewa KL, Motrescu ER, Chenard MP, Gansmuller A, Stoll I, Gregoire FM. (2001). Adipocyte differentiation: from fibroblast to Tomasetto C et al. (2005). Stromelysin-3 is a potent negative endocrine cell. Exp Biol Med (Maywood) 226: 997–1002. regulator of adipogenesis participating to cancer cell-adipocyte Han J, Daniel JC. (1995). Biosynthesis of type VI collagen by interaction/crosstalk at the tumor invasive front. Cancer Res 65: glioblastoma cells and possible function in cell invasion of three- 10862–10871. dimensional matrices. Connect Tissue Res 31: 161–170. Basset P, Bellocq JP, Wolf C, Stoll I, Hutin P, Limacher JM Iyengar P, Combs TP, Shah SJ, Gouon-Evans V, Pollard JW, et al. (1990). A novel metalloproteinase specifically Albanese C et al. (2003). Adipocyte-secreted factors synergistically expressed in stromal cells of breast carcinomas. Nature 348: promote mammary tumorigenesis through induction of anti- 699–704. apoptotic transcriptional programs and proto-oncogene stabiliza- Basset P, Okada A, Chenard MP, Kannan R, Stoll I, Anglard P et al. tion. Oncogene 22: 6408–6423. (1997). Matrix as stromal effectors of human Iyengar P, Espina V, Williams TW, Lin Y, Berry D, Jelicks LA carcinoma progression: therapeutic implications. Matrix Biol 15: et al. (2005). Adipocyte-derived collagen VI affects early 535–541. mammary tumor progression in vivo, demonstrating a critical Bernlohr DA, Angus CW, Lane MD, Bolanowski MA, Kelly Jr TJ. interaction in the tumor/stroma microenvironment. J Clin Invest (1984). Expression of specific mRNAs during adipose 115: 1163–1176. differentiation: identification of an mRNA encoding a Kannan R, Ruff M, Kochins JG, Manly SP, Stoll I, El Fahime M et al. homologue of myelin P2 protein. Proc Natl Acad Sci USA 81: (1999). Purification of active matrix metalloproteinase catalytic 5468–5472. domains and its use for screening of specific stromelysin-3 inhibitors. Boulay A, Masson R, Chenard MP, El Fahime M, Cassard L, Bellocq Protein Expr Purif 16: 76–83. JP et al. (2001). High cancer cell death in syngeneic tumors Kielty CM, Lees M, Shuttleworth CA, Woolley D. (1993). Catabolism developed in host mice deficient for the stromelysin-3 matrix of intact type VI collagen microfibrils: susceptibility to degradation metalloproteinase. Cancer Res 61: 2189–2193. by serine proteinases. Biochem Biophys Res Commun 191: Celis JE, Moreira JM, Cabezon T, Gromov P, Friis E, Rank F et al. 1230–1236. (2005). Identification of extracellular and intracellular signaling Kuo HJ, Maslen CL, Keene DR, Glanville RW. (1997). Type VI components of the mammary adipose tissue and its interstitial fluid collagen anchors endothelial basement membranes by interacting in high risk breast cancer patients: toward dissecting the molecular with type IV collagen. J Biol Chem 272: 26522–26529. circuitry of epithelial-adipocyte stromal cell interactions. Mol Cell Lamande SR, Morgelin M, Adams NE, Selan C, Allen JM. (2006). Proteomics 4: 492–522. The C5 domain of the collagen VI alpha3(VI) chain is critical for

Oncogene Collagen VI is a substrate of MMP11 ER Motrescu et al 6355 extracellular microfibril formation and is present in the Noel A, Boulay A, Kebers F, Kannan R, Hajitou A, Calberg-Bacq extracellular matrix of cultured cells. J Biol Chem 281: CM et al. (2000). Demonstration in vivo that stromelysin-3 functions 16607–16614. through its proteolytic activity. Oncogene 19: 1605–1612. Manabe Y, Toda S, Miyazaki K, Sugihara H. (2003). Mature Noel A, Santavicca M, Stoll I, L’Hoir C, Staub A, Murphy G et al. adipocytes, but not preadipocytes, promote the growth of breast (1995). Identification of structural determinants controlling human carcinoma cells in collagen gel matrix culture through cancer- and mouse stromelysin-3 proteolytic activities. J Biol Chem 270: stromal cell interactions. J Pathol 201: 221–228. 22866–22872. Masson R, Lefebvre O, Noel A, Fahime ME, Chenard MP, Wendling Pei D, Majmudar G, Weiss SJ. (1994). Hydrolytic inactivation of a C et al. (1998). In vivo evidence that the stromelysin-3 metallopro- breast carcinoma cell-derived serpin by human stromelysin-3. J Biol teinase contributes in a paracrine manner to epithelial cell Chem 269: 25849–25855. malignancy. J Cell Biol 140: 1535–1541. Pei D, Weiss SJ. (1995). -dependent intracellular activation of the McCawley LJ, Matrisian LM. (2001). Matrix metalloproteinases: human stromelysin-3 zymogen. Nature 375: 244–247. they’re not just for matrix anymore. Curr Opin Cell Biol 13: Rio MC. (2002). Stromelysin-3, a Particular Member of the Matrix 534–540. Metalloproteinase Family, kluwer academic edn. Kluwer Academic Motrescu ER, Rio MC. (2008). Cancer cells, adipocytes and matrix Publisher: Dordrecht. metalloproteinase 11: a vicious tumor progression cycle. Biol Chem 389. Ruhl M, Johannsen M, Atkinson J, Manski D, Sahin E, Soma- Myint E, Brown DJ, Ljubimov AV, Kyaw M, Kenney MC. (1996). sundaram R et al. (1999). Soluble collagen VI induces tyrosine Cleavage of human corneal type VI collagen alpha 3 chain by matrix phosphorylation of paxillin and focal adhesion kinase and activates metalloproteinase-2. Cornea 15: 490–496. the MAP kinase erk2 in fibroblasts. Exp Cell Res 250: 548–557. Nakajima I, Muroya S, Tanabe R, Chikuni K. (2002). Positive effect of Schaffler A, Scholmerich J, Buechler C. (2007). Mechanisms of disease: collagen V and VI on triglyceride accumulation during differentia- adipokines and breast cancer—endocrine and paracrine mechanisms tion in cultures of bovine intramuscular adipocytes. Differentiation that connect adiposity and breast cancer. Nat Clin Pract Endocrinol 70: 84–91. Metab 3: 345–354.

Oncogene