Role and Function of Matrix Metalloproteinases ... - Wiley Online Library

THE STEM CELL NICHE: CONCISE REVIEW Role and Function of Matrix Metalloproteinases in the Differentiation and Biological Characterization of Mesenchymal Stem Cells FERDINANDO MANNELLO,a GAETANA A.M. TONTI,a GIAN PAOLO BAGNARA,c,d STEFANO PAPAb a

Institute of Histology and Laboratory Analysis and bInstitute of Morphological Sciences, Center of Cytometry, University “Carlo Bo” of Urbino, Urbino, Italy; cDepartment of Histology, Embryology, and Applied Biology and d Stem Cell Research Center, University of Bologna, Bologna, Italy Key Words. Mesenchymal stem cell • Matrix metalloproteinase • Tissue inhibitor of metalloproteinases Gene and protein expression • Differentiation • Osteogenesis • Chondrogenesis • Adipogenesis Neurogenesis • Myogenesis

ABSTRACT Matrix metalloproteinases (MMPs), known as matrixins, are Ca- and Zn-dependent endoproteinases involved in a wide variety of developmental and disease-associated processes, proving to be crucial protagonists in many physiological and pathological mechanisms. The ability of MMPs to alter, by limited proteolysis and through the fine control of tissue inhibitors of metalloproteinases, the activity or function of numerous proteins, enzymes, and receptors suggests that they are also involved in various important cellular functions during development. In this review, we focus on the differentiation of

mesenchymal stem cells (including those of the myoblastic, osteoblastic, chondroblastic, neural, and apidoblastic lineages) and the possible, if unexpected, biological significance of MMPs in its regulation. The MMP system has been implicated in several differentiation events that suggests that it mediates the proliferative and prodifferentiating effect of the matrixin proteolytic cascade. We summarize these regulatory effects of MMPs on the differentiation of mesenchymal stem cells and hypothesize on the function of MMPs in the stem cell differentiation processes. STEM CELLS 2006;24:475– 481

INTRODUCTION

[4], MSCs have up to now, been isolated from other tissue sources, suggesting that MSCs are diversely distributed in vivo and may occupy an ubiquitous stem cell niche [5]. The heterogeneity of MSCs, demonstrated in both in vivo and in vitro studies [2], with respect to their self-renewal and differentiation potentials, can be explained by the notion that the MSC pool comprises not only putative “mesenchymal stem cells” but also subpopulations at different states of differentiation (e.g., quadra-, tri-, bi-, and unipotential MSCs) [5]. Depending on the specific culture conditions and stimuli used, MSCs are able to form bone, cartilage, tendon, muscle, fat, and neural tissue as well as hematopoietic-supporting stroma [3]. MSCs can also be transdifferentiated by specific transcriptional activators, even though it is not understood what specific environmental cues are necessary to initiate their proliferation and differentiation, producing autocrine and paracrine factors essential for lineage progression [1, 6]. Despite increasing biomolecular and morphologic knowledge of MSCs, an understanding of the full

Adult Mesenchymal Stem Cells Mesenchymal stem cells (MSCs) have generated a great deal of excitement and expectation as a potential source of cells for cell-based therapeutic strategies, primarily because of their intrinsic ability to self-renew and differentiate into functional cell types that constitute the tissue in which they exist [1]. Despite diverse and ever-growing information concerning MSCs and their use in clinical strategies, the mechanisms that govern MSC self-renewal and multilineage differentiation are not well understood and remain an active area of investigation [2]. Therefore, research efforts focused on identifying factors that regulate and control MSC fate decisions become crucial in the promotion of a greater understanding of the molecular, biological, and physiological characteristics of this potentially highly useful stem cell type [3]. Despite the fact that the pioneering experimental evidence supports the existence of bone marrow– derived MSCs

Correspondence: Ferdinando Mannello, Ph.D., Istituto di Istologia ed Analisi di Laboratorio, Facolta` di SMFN, Universita` Studi “Carlo Bo,” Via E. Zeppi, 61029 Urbino (PU), Italy. Telephone: ⫹39-0722-351479; Fax: 39-0722-322370; e-mail: f.mannello@ uniurb.it; Web site: http://www.uniurb.it/istoanalab/index.htm Received July 24, 2005; accepted for publication September 2, 2005; first published online in STEM CELLS EXPRESS September 8, 2005. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0333

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functional differentiation capacity of MSCs, the mechanisms controlling their mobilization and their homing properties, their differentiation program to various tissue types, and their physiological role are needed. In this respect, several studies have provided evidence on the importance of the expression and production of both adhesion molecules and extracellular matrix (ECM) components [2, 3], which contribute to the formation and function of a unique microenvironment that produces induction-regulating signals [6, 7]. Many adhesion molecules and ECM proteins identified in MSCs are regulated/activated and made biologically functional through proteolysis by matrix metalloproteinases (MMPs), endopeptidases belonging to the family of matrixins that are able to regulate several physiological processes [8]. The interaction between MMP proteolytic activity and the multifaceted functions of tissue inhibitors of metalloproteinases (TIMPs) are biologically crucial for many developmental events [9], including morphogenesis, cell proliferation, and apoptosis [10, 11], as well as tissue development, through the modulation of biologically active molecules [12, 13]. Recently, biomolecular studies highlighted, in MSCs, the gene expression involved in the connection between cell-matrix and cell-cell external signals, as well as in the intracellular signalling pathways. In fact, MMP [14 –16] and TIMP [14, 15] genes have been identified in MSCs derived from different biological sources, indicating both their common ontogeny and the activation of similar sets of genes because of their close functional roles. Their gene-expression profiles, as part of the transcriptome of MSCs, may provide possible explanations for MSC functioning and behavior.

MMPs and TIMPs MMPs are a family of Ca- and Zn-dependent endopeptidases with the capacity to cleave most of the ECM components, expressed ubiquitously or in a tissue-specific way as intracellular and intranuclear enzymes [11]. They are distributed within the animal kingdom from mammals to invertebrates [17]. More than 25 enzymes have been identified to date and, according to their substrate specificity, they have been divided into collagenases, gelatinases, stromelysins, membrane-type (MT-MMP), and other less well characterized types [18]. All MMPs possess a catalytic domain containing the Zn-binding site, which is maintained inactive through the interaction of a cysteine residue in the propeptide with the catalytic Zn. MMPs may be secreted or may be transmembrane proenzymes that acquire full activity after the removal of the N-terminal propeptide, which disrupts the interaction between the Zn ion and the cysteine residue [18]. Activation through the “cysteine switch” allows for the regulation of the level and cellular compartmentalization of the enzymes, as well as for interaction with the four endogenous TIMPs. TIMPs are tissue-specific inhibitors of matrixins that participate in controlling the local activities of MMPs, and their expression is highly regulated during development and tissue remodeling [10, 12, 18]. In addition to MMP-inhibiting activities, TIMPs have other biological functions (such as erythroidpotentiating activity, cell growth–promoting activity, and mesenchymal growth-regulating capability), activities not attributed to MMP inhibition that remain largely to be discovered [10]. Their possible translocation to the nucleus allows TIMPs to participate in the programmed cell death machinery, even though with apparently contrasting roles [11].

MMPs and TIMPs During MSC Differentiation MMPs were originally referred to as intracellular or transmembrane enzymes that regulate the activity of many biological molecules by cleaving or releasing them [13]; however, it has also been recognized that MMPs can undergo specific subcellular relocation from the membrane or the cytosol to the nucleus, where they may proteolyze transcription factors and nuclear proteins, playing discordant roles in both cell proliferation and apoptosis [10, 11]. These enzymes and their specific inhibitors represent multipotent effectors with a huge activity spectrum in cellular development and physiology. In addition to the well-known roles and functions of MMPs and TIMPs in many disease processes involving both ECM and nuclear matrix degradation and remodeling (e.g. inflammatory and degenerative diseases, and cancer) [8, 12], these multifunctional proteins play an essential role during embryonic development, morphogenesis, and cell and tissue developmental processes, through cleavage and release of ECM microenvironment molecules and modulation of general or tissue-specific gene expression [9]. Moreover, several studies suggest that MMPs and TIMPs can affect fundamental cellular processes (such as proliferation, survival, and apoptosis), key processes of cell differentiation [11].

ROLE AND FUNCTION OF MMPS DURING DIFFERENTIATION

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TIMPS

Myogenesis Modifications of the ECM environment and the breakdown of connective tissue barriers are necessary and fundamental events in the differentiation, development, and regeneration of myoblasts and the stem cell replenishment of muscle that require cell migration and fusion [19]. Many studies have provided evidence that myoblasts migrate within and between adult muscles during muscle regeneration [20] and have shown the presence of myogenic stem cells outside the muscle that can potentially contribute to muscle growth and repair. Moreover, pluripotent cells with myogenic potential have been found in many nonmuscle tissues, and in order to contribute to myogenesis, these cells must migrate through the muscle and cross the basal lamina [21]. Although the exact role of MMPs during myoblast migration in vivo is not yet clear, in vitro studies have provided evidence of their important functions during myogenetic events. The transcripts of MMP-2 and MT1-MMP were proven to be essential to the high migratory capacity of the C2C12 murine myoblast cell line; this was also demonstrated by the inhibition of muscle development and regeneration observed in vivo using the specific synthetic MMP inhibitor batimastat [22]. Moreover, human skeletal muscle satellite cells constitutively express MMP-2, but under certain treatment conditions (e.g., induction by phorbol ester), these cells may also express MMP-9, suggesting that satellite cells can synthesize and secrete specific MMPs (high level of both MMP-2, MMP-9 and little or no collagenase MMP-1 or stromelysin MMP-3), taking part in the myogenesis and skeletal muscle regeneration through remodeling of the ECM [23]. MMP-9 was expressed only in single-cell and prefusion in vitro muscle cultures, whereas MMP-2 was secreted during all stages of myoblast migration and differentiation, as well as by myotubes. Moreover, TIMP-1 expression (absent during the single-cell stage) increased during the prefusion and postfusion stages, reaching maximal expression during myotube formation in the C2C12 cell line [19].

Mannello, Tonti, Bagnara et al. The involvement of MMPs in the fusion of muscle cells was demonstrated by the high fusion index of a myoblast cell line expressing MMP-7. In particular, matrilysin MMP-7 expression may be useful in increasing myoblast transplantation success, improving the myogenic potential of myoblasts in vitro and the fusion of myoblasts with host fibers in vivo [24]. Myoblast differentiation, migration, and invasion are, in part, a result of increased MMP activity, which has been positively modulated by several growth factors and ECM proteins [25]. In fact, whereas fibronectin, basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor ␤ 1 (TGF-␤1) significantly enhance migration of mouse myoblasts, tumor necrosis factor ␣, platelet-derived growth factor, and insulin growth factor 1 do not. These observations support the hypothesis that MMP modulation is a necessary component of growth factor–mediated myoblast signalling [25]. Moreover, increased MMP-9 activity has been found in adult skeletal muscle injury and in myopathy, suggesting that this MMP may be involved in muscle regeneration and repair [26, 27]. Although there is not a direct connection between growth factor stimulation/ECM protein secretion and MMP activity, it is clear that MMPs and their inhibitors, TIMPs, are essential in ruling myogenesis, myoblast differentiation, and the migration process.

Neurogenesis The first reports suggesting that MMPs may play important and unexpected roles during neuronal development took into consideration the embryonic development of the mouse brain. In particular, high MMP-9 expression levels were found in progenitor cells associated with the development of specific structures, such as the hypophysis, choroid plexus, ganglion cell layer of the retina, and uveal tract, and in aggregates that would later form the highly vascular grey matter of the brain [28]. Even though, in the adult central nervous system (CNS), most MMPs are expressed at very low levels and their upregulation has been associated with detrimental effects in several neurological disorders and linked to repair after injury [29], recent reports highlight the beneficial properties of many MMPs during CNS development [30, 31]. Because of their role in modulating the motility of cells across tissue matrices, it is possible that MMPs may also regulate the migration of precursor stem cells to their final destination during neuronal development [29]. The expression of MMP-2, but not MMP-9, and all TIMPs has been found in neuroepithelial stem cells isolated from the human CNS. In particular, low expression levels of both MMP-2 and TIMP-4 were found in mature CNS cells, with higher concentrations in stem cells. Moreover, while the expression levels of TIMP-1, TIMP-2, and TIMP-3 were unchanged following stem cell differentiation into neurons and glia, the high expression level of TIMP-4 in neuroectodermal precursor cells was associated with stem cells, in which its molecular level remained unchanged upon differentiation [32]. Moreover, MMP-9 seems to be involved during myelinogenesis, because the extension of oligodendroglial processes could require remodeling of the brain matrix, while its inhibition blocked the expansion of processes from the cell soma [33]. High levels of MMP-12 transcripts in microglia [34] and also in oligodendrocytes [35] were correlated with process extension, suggesting a role in the maturation and morphological differentiation of precursor stem cells [35]. Several inducers of both neuronal www.StemCells.com

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differentiation and axonal growth increased MMP-2, MMP-3 and MMP-9 expression levels, suggesting their involvement both in axon elongation and neurite growth [36, 37], probably through the degradation or inactivation of the neurite-inhibiting activity of nerve proteoglycans [38]. It has been hypothesized that MMPs may also be involved in injury repair, favoring the migration of precursor stem cells to injured sites to replenish lost cells and thus contributing to axonal regrowth, remyelination, and angiogenesis [29]. Moreover, it was demonstrated that reactive astrocytes are involved in the neurodegeneration/repair response in wobbler mice, because MT1-MMP and TIMP-1 and TIMP-3 levels were elevated in brain stem cells and the spinal cord during neuronal regeneration [39]. In conclusion, MMPs and TIMPs may play important regulatory roles during differentiation of neural/neuronal precursor cells, suggesting unexpected roles in stem cell development.

Chondrogenesis Proteolytic remodeling of the ECM is an important event during skeletal formation, strictly regulated at both the temporal and spatial levels, requiring chondrocytic cells that undergo continuous differentiation processes. During endochondral ossification in mice, chondrocytes of the lower zone of hypertrophic cartilage expressed MMP-13 together with type X collagen at specific embryonic stages, suggesting differentiation-dependent expression of this collagenase [40]. Even though several MMPs are involved and expressed at higher levels during pathological conditions (e.g., rheumatoid arthritis) [8, 12], tissue cultures of human healthy cartilage showed the production of stromelysin MMP-3 during chondrocyte-mediated remodeling events of the cartilage [41]. In addition, MMPs have also been detected during bone formation and remodeling [42]. In cartilage rudiment cultures and in primary chondrocyte cultures undergoing endochondral ossification, the expression of MMP-13 was increased by treatment with retinoic acid (RA, an effector of several events during bone differentiation) [43]. RA regulates the expression of MMP-13 through the p38 mitogenactivated protein kinase pathway during the osteogenic differentiation process [43]. In the same way, MT1-MMP is also involved in skeletal formation [44]. Because the addition of RA to chondrocytic cells induced morphological changes, the expression of MMP-13 and MT1-MMP may reflect defined stages of the differentiation process, suggesting their crucial roles in replacing cartilage with bone during development [43]. It has been hypothesized that the wide substrate specificity of MMP-13 may support ECM degradation to favor mature bone formation, and in association with other MMPs, it may release growth factors sequestered or blocked in the matrix scaffold [45]. In addition to results showing that the activity of MT1MMP is physiologically crucial for adequate collagen turnover, mice deficient in this enzyme also develop severe connective tissue disease [46]. Reports analyzing the in vitro differentiation pathway of chondrocyte-like cells and in vivo cartilage repair processes have highlighted the role of MT1-MMP in regulating pericellular proteolysis, either directly or through the involvement of other MMPs (such as MMP-2, MMP-9, and MMP-13) [47]. MMP-9 may be involved in correct endochondral bone formation, probably because of its capacity to degrade type II, IX, and XI collagen, activating procollagenase I and releasing

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angiogenic factors (such as vascular endothelial growth factor from the hypertrophic cartilage ECM) [48], ultimately leading to an essential role in chondrocytic differentiation pathways. Because of their degradative properties, MMPs may also be involved in the induction of chondrocytic chondrolysis in inflammatory and degenerative joint diseases, suggesting a physiological role in the degradation of the cartilage matrix and in differentiation pathways of chondrocytic stem cells [49]. Hence, MMPs may play a central role in chondrogenesis, promoting chondrocyte terminal maturation and representing signalling molecules that mediate the chondrogenic differentiation program [50]. Moreover, TGF-␤–induced chondrogenesis signalling pathways may be regulated by both the extracellular signal– related kinase and MMP signalling cascades. The functional potential of these differentiation mechanisms may emphasize the involvement of MMPs in the regulation of chondrogenesis from human MSCs, leading to innovative protocols for clinical orthopedic interventions [51].

Osteogenesis The differentiation and proliferation of osteoblast progenitors is a complex process that requires continuous ECM remodeling and the interaction of numerous hormones, autocrine and paracrine processes, and systemic growth factors. Matrix elaboration and modification are fundamental in osteoclast-dependent bone resorption, and MMPs are necessary for the migration of precursor and immature osteoclasts to the bone surface [52]. In undifferentiated MSCs, many cancer testis genes (e.g., SSX, NY-ESO-1, N-RAGE) are expressed and become downregulated after osteocyte differentiation; during osteogenesis MSCs show a concomitant reduction in MMP-2 levels, suggesting a role in stem cell migration [53]. Studies on in vivo transplantation systems showed that bone marrow stromal stem cells express high levels of basic fibroblast growth factor and MMP-9 that correlate with the formation of hematopoietic marrow in bone marrow stromal stem cell transplants, suggesting the involvement of MMPs in stem-cell-mediated bone regeneration [54]. As osteocytes are the result of the final differentiation step of osteogenic mesenchymal progenitors (immobilized in the bone matrix, interacting with other cells through extension of cell processes), it is feasible that osteocytes actively interact with their ECM environment and in particular with type I collagen, which is the most abundant protein in the osteocyte environment. Both collagenolytic activity and osteocyte process extension have been demonstrated to be dependent on MT1MMP expression, which is necessary for both osteocytogenesis and the maintenance of the osteocyte phenotype [55]. Other than being linked to the differentiation of MSCs into osteocytes, MMPs are also involved in the regulation of osteoblast proliferation and apoptosis. In fact, it has been hypothesized that MT1-MMP, produced by osteoblasts through the activation of latent TGF-␤, is involved in cell survival during transdifferentiation into osteocytes [56] and favors mature osteocyte viability through insensitivity to MMP inhibitors [57]. The importance of MMPs in osteoclastic resorption has been supported by studies showing that MMP inhibitors (such as tetracyclines and their modified derivatives) [12] may block the in vitro osteoclastogenesis of human peripheral blood mononuclear cells stimulated with M-CSF [58].

MMPs and TIMPs During MSC Differentiation Activation of parathyroid hormone receptors in osteoblasts has been shown to increase the expression levels of several MMPs (MMP-9, MMP-13) through increased stimulation of the phospholipase C pathway, leading to upregulated expression of the AP-1 factors c-jun and c-fos [59]. MMPs are thought to also play a role in the bone remodeling and transdifferentiation balance. In fact, studies on mice overexpressing TIMP–1 have provided evidence that the MMP/TIMP ratio is highly regulated during osteogenesis and is probably involved in the hormonal control of bone remodeling by osteoblasts [60]. In vitro studies have suggested that the transdifferentiation of MSCs and/or the function of bone marrow stromal cells depend on both MMP activities and TIMP inhibitory functions, which are capable of regulating the development and proliferation of these cells [54, 61].

Adipogenesis The development of the fat mass consists of adipocyte hypertrophy and hyperplasia resulting from the recruitment and differentiation of preadipocytes into adipocytes. Moreover, for correct growth of the fat depot, angiogenesis and ECM proteolytic remodeling are also of great importance [62]. Formation of adipose tissue involves the commitment of mesodermal/mesenchymal cells to a preadipocyte lineage and differentiation of preadipocytes into mature adipocytes; differentiation correlates with increased secretion of and matrixin-dependent degradation of both basement membrane and ECM components [63]. Animal studies have shown that, during adipocyte differentiation, there is breakdown and remodeling of the original ECM through the secretion of MMP-2 [64] and reduced expression of TIMP-1 [65]. Noteworthy is the fact that the stromelysin MMP-3 is expressed by fibroblastic cells, some of which are preadipocytes [66], and that it is activated in parallel with markers usually linked to active remodeling and morphogenesis [67]. In fact, in adipogenic cells, mRNA of MMP-3, MT1-MMP, and MMP-13 is induced in committed preadipocytes, underlining the fact that the transition from preadipocytes to terminally differentiated fat cells occurs in parallel with increased matrixin proteolytic activity and concomitant with TIMP-1 and TIMP-3 downregulation [67]. Adipocytes differ from their mesenchymal precursors because of increased accumulation of basement membrane, which may lead to terminal differentiation by stabilizing the adipocyte cell surface and generating specific intracellular signals. The assembly of ECM may be the rate-limiting step in preadipocyte differentiation, so the secretion of MMPs may control/limit their development; in fact, the addition of MMP inhibitors during the onset of differentiation strongly accelerates lipogenesis by blocking the MMP-dependent degradation of entactin and inhibiting the ECM assembly [67]. MMP-2 and MMP-9 are also important regulators of adipocyte differentiation. Specifically, MMP-2 is secreted from undifferentiated preadipocytes and increases along with the differentiation process, while MMP-9, not present in undifferentiated preadipocytes, strongly increases its activity during adipocyte differentiation stages [68]. It has been hypothesized that MMP-9 affects adipogenesis through the regulation of adipogenic cytokines (e.g., interleukin [IL]-8, IL-1␤, IL-6) [69]. In vitro studies on adipocyte differentiation have shown that committed cells presented increased levels of MMP-2 and MMP-9, concomitantly with the disappearance of TIMP-1 when preadipocytes started

Mannello, Tonti, Bagnara et al. the differentiation processes [68]. In the final differentiation stage, adipocytes also present increasing levels of mRNA for MMP-19 [70]. All the MMP and TIMP modulations reveal the crucial role that they may play during fat cell differentiation and conversion.

FUTURE PERSPECTIVES FOR THE BIOLOGICAL CHARACTERIZATION OF MSCS MMPs and TIMPs play essential positive and negative roles in the differentiation of chondroblasts, osteoblasts, and adipoblasts, which are derived from adult common MSCs, even though these proteins seem to also be involved in the commitment of myoblasts and neuronal/glial cells. MMPs may be involved in the early stage of the differentiation of all cell lineages and may relocate to plasma membranes or nuclear fractions. A vast amount of work remains to be done in the field of MSC differentiation. In particular, several pieces of evidence demonstrate that uncommitted MSCs contain the biomolecular machinery of MMP and TIMP expression; as well, differentiated cells show a characteristic distribution of MMPs and TIMPs. Figure 1 depicts what happens to MMPs and TIMPs during MSC differentiation. Although out, understanding of the promoter and enhancer regions of MMP genes during MSC differentiation is still in its infancy [14, 16], a great number of factors can stimulate or repress matrixin proteolytic expression and upregulate or downregulate TIMP function, leading to the following questions. How are the effects of MMPs and TIMPs

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produced on the genes in the stem and/or committed cell compartment? Why and how is ECM so intimately linked with this expression? Are MMPs to be expected in the activated form or, if not, what mechanisms exist for the activation of MMP enzymes? During MSC differentiation, do MMP protein domains remain unmodified or undergo structural alterations (e.g., the addition or loss of domains originally found in the ancestral MMPs)? Does protein modification influence the functional activity and substrate specificity of MMPs during the different differentiation states? Do increased MMP and TIMP activities play a role in the dedifferentiation of fully committed cells? Does the MMP/TIMP balance modulate the predetermined cell fate of MSCs? Although this unexplored field of research produces more questions than answers, the MMP/TIMP balance may represent a sophisticated vision of specific proteolytic events during MSC differentiation, targeting both matrix and nonmatrix substrates driving cellular proliferation, apoptosis, and development. However, there is a paucity of information about the effects of MMPs and TIMPs on the transcriptional factors that function in the differentiation of MSC-derived blasts, even though consistent evidence underlines the significant relationship between MMPs and the ECM during differentiation. This opens up new frontiers on the modulation of ECM components through the production of MMPs and their fine control by TIMPs as important key regulators during MSC differentiation. The MMP/TIMP system thus plays important roles in cell proliferation [11, 13] and differ-

Figure 1. Involvement of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the differentiation hierarchy of human adult mesenchymal stem cells (MSCs). The scheme depicts the three compartments of adult MSC differentiation: the uncommitted multipotent MSC has the potential for self-renewal and to give rise to a less potent precursor cell population via asymmetric cell division; the committed mesenchymal progenitors cells show a lower level of stemness and a more restricted differentiation potential, via symmetric cell division. The uniand tripotent precursor cells generate, via symmetric division, cells with predetermined cell fates, progenitors named colony-forming unit (CFUs). These cells give rise to fully committed/differentiated/mature mesenchymal phenotypes that may, under particular conditions, dedifferentiate into more potent cells with a higher level of stemness. During adult MSC differentiation, transcriptional and phenotypic modifications increase, whereas proliferative potential progressively decreases, concomitantly with limited multilineage potential. It is known that several MMPs and TIMPs play crucial roles and functions during the terminal stages of MSC progenitor differentiation into mature phenotypes. Because molecular studies have provided evidence that both MMP and TIMP genes are present in multipotent MSCs, the MMP/TIMP balance may play a key role in self-renewal as well as in the precursor and progenitor differentiation of human adult MSCs. Abbreviations: O, osteocyte; C, chondrocyte; A, adipocyte; sk, skeletal, sm, smooth muscle cell; c, cardiac muscle cell; As, astrocyte; Ol, oligodendrocyte; N, neuron.

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entiation [9, 61] in many tissues. The MMP/TIMP balance may be a crucial factor in the control of MMP activity in vivo [10]. In fact, this balance regulates the amounts of several biologically active cellular components that participate in MSC differentiation, even though it is not exactly clear how the MMP/ TIMP system works in the different stages of differentiation. In this respect, the identification and characterization of the biomolecular expression of both MMPs and TIMPs in MSCs isolated from different biological sources (umbilical cord vein, placental and fetal membranes, and dental pulp) are currently in

REFERENCES 1

Barry FP, Murphy JM. Mesenchymal stem cells: Clinical applications and biological characterization. Int J Biochem Cell Biol 2004;36:568 – 584.

MMPs and TIMPs During MSC Differentiation progress (F. Mannello et al., in preparation). Understanding how the various biologically active molecules are upregulated or downregulated by the MMP/TIMP balance in the course of MSC differentiation remains an important area for future research and may lead to the development of novel therapeutic approaches against diseases that involve stem cell differentiation.

DISCLOSURES The authors indicate no potential conflicts of interest.

20 Philips GD, Hoffman JR, Knighton DR. Migration of myogenic cells in rat extensor digitorum longus muscle studied with a split autograft model. Cell Tissue Res 1990;262:81– 88. 21 Galli R, Borello U, Gritti A et al. Skeletal myogenic potential of human and mouse neural stem cells. Nat Neurosci 2000;3:986 –991.

2

Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med (Maywood) 2001;226:507–520.

3

Roufosse CA, Direkze NC, Otto WR et al. Circulating mesenchymal stem cells. Int J Biochem Cell Biol 2004;36:585–597.

4

Friedenstein AJ. Osteogenic activity of transplanted transcriptional epithelium. Acta Anat (Basel) 1961;45:31–59.

23 Guerin CW, Holland PC. Synthesis and secretion of matrix-degrading metalloproteases by human skeletal muscle satellite cells. Dev Dyn 1995;202:91–99.

5

Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: Characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 2004;8:301–316.

24 Caron NJ, Asselin I, Morel G et al. Increased myogenic potential and fusion of matrilysin-expressing myoblasts transplanted in mice. Cell Transplant 1999;8:465– 476.

6

Bianco P, Riminucci M, Gronthos S et al. Bone marrow stromal stem cells: Nature, biology, and potential applications. STEM CELLS 2001; 19:180 –192.

25 Allen DL, Teitelbaum DH, Kurachi K. Growth factor stimulation of matrix metalloproteinase expression and myoblast migration and invasion in vitro. Am J Physiol Cell Physiol 2003;284:C805–C815.

7

Zipori D. Mesenchymal stem cells: Harnessing cell plasticity to tissue and organ repair. Blood Cells Mol Dis 2004;33:211–215.

8

Nelson AR, Fingleton B, Rothenberg ML et al. Matrix metalloproteinases: Biologic activity and clinical implications. J Clin Oncol 2000;18: 1135–1149.

9

Vu TH, Werb Z. Matrix metalloproteinases: Effectors of development and normal physiology. Genes Dev 2000;14:2123–2133.

10 Mannello F, Gazzanelli G. Tissue inhibitors of metalloproteinases and programmed cell death: Conundrums, controversies and potential implications. Apoptosis 2001;6:479 – 482. 11 Mannello F, Luchetti F, Falcieri E et al. Multiple roles of matrix metalloproteinases during apoptosis. Apoptosis 2005;10:19 –24. 12 Mannello F, Tonti G, Papa S. Matrix metalloproteinase inhibitors as anticancer therapeutics. Curr Cancer Drug Targets 2005;5:285–298. 13 Mott JD, Werb Z. Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 2004;16:558 –564. 14 Silva WA Jr, Covas DT, Panepucci RA et al. The profile of gene expression of human marrow mesenchymal stem cells. STEM CELLS 2003;21:661– 669. 15 Panepucci R, Siufi JL, Silva WA Jr et al. Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells. STEM CELLS 2004;22:1263–1278. 16 Steck E, Bertram H, Abel R et al. Induction of invertebral disc-like cells from adult mesenchymal stem cells. STEM CELLS 2005;23:403– 411. 17 Mannello F, Tonti G, Papa S. Are matrix metalloproteinases the missing link? ISJ 2005;2:69 –74. Available at http://www.isj.unimo.it/vol2issue1. htm. Accessed May 30, 2005.

22 El Fahime E, Torrente Y, Caron NJ et al. In vivo migration of transplanted myoblasts requires matrix metalloproteinase activity. Exp Cell Res 2000;258:279 –287.

26 Roach DM, Fitridge RA, Laws PE. Up-regulation of MMP-2 and MMP-9 leads to degradation of type IV collagen during skeletal muscle reperfusion injury; protection by the MMP inhibitor, doxycycline. Eur J Vasc Endovasc Surg 2002;23:260 –269. 27 Schoser BG, Blottner D, Stuerenburg HJ. Matrix metalloproteinase in inflammatory myopathies: Enhanced immunoreactivity near atrophic myofibers. Acta Neurol Scand 2002;105:309 –313. 28 Canete Soler R, Gui YH, Linask KK et al. MMP-9 (gelatinase B) mRNA is expressed during mouse neurogenesis and may be associated with vascularization. Brain Res Dev Brain Res 1995;88:37–52. 29 Yong VW, Power C, Forsyth P et al. Metalloproteinases in biology and pathology of the nervous system. Nat Rev Neurosci 2001;2:502–511. 30 Vaillant C, Didier-Bazes M, Hutter A et al. Spatiotemporal expression patterns of metalloproteinases and their inhibitors in the postnatal developing rat cerebellum. J Neurosci 1999;19:4994 –5004. 31 Sekine-Aizawa Y, Hama E, Watanabe K et al. Matrix metalloproteinase (MMP) system in brain: Identification and characterization of brainspecific MMP highly expressed in cerebellum. Eur J Neurosci 2001;13: 935–948. 32 Frolichsthal-Schoeller P, Vescovi AL, Krekoski CA et al. Expression and modulation of matrix metalloproteinase-2 and tissue inhibitors of metalloproteinases in human embryonic CNS stem cells. Neuroreport 1999; 10:345–351. 33 Oh LY, Larsen PH, Krekoski CA et al. Matrix metalloproteinase-9/ gelatinase B is required for process outgrowth by oligodendrocytes. J Neurosci 1999;19:8464 – 8475. 34 Shapiro SD, Kobayashi DK, Ley TJ. Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages. J Biol Chem 1993;268:23824 –23829.

18 Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases. Circ Res 2003;92:827– 839.

35 Larsen PH, Yong VW. The expression of matrix metalloproteinase-12 by oligodendrocytes regulates their maturation and morphological differentiation. J Neurosci 2004;24:7597–7603.

19 Lewis MP, Tippett HL, Sinnan AC et al. Gelatinase-B (matrix metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures. J Muscle Res Cell Motil 2000; 21:223–233.

36 Chambaut-Guerin AM, Herigault S, Rouet-Benzineb P et al. Induction of matrix metalloproteinase MMP-9 (92-kDa gelatinase) by retinoic acid in human neuroblastoma SKNBE cells: Relevance to neuronal differentiation. J Neurochem 2000;74:508 –517.

Mannello, Tonti, Bagnara et al.

37 Zuo J, Ferguson TA, Hernandez YJ et al. Neuronal matrix metalloproteinase-2 degrades and inactivates a neurite-inhibiting chondroitin-sulphate proteoglycan. J Neurosci 1998;18:5203–5211. 38 Webber CA, Hocking JC, Yong VW et al. Metalloproteases and guidance of retinal axons in the developing visual system. J Neurosci 2002; 22:8091– 8100. 39 Rathke-Hartlieb S, Budde P, Ewert S et al. Elevated expression of membrane type 1 metalloproteinase (MT1-MMP) in reactive astrocytes following neurodegeneration in mouse central nervous system. FEBS Lett 2000;481:227–234. 40 Tuckermann JP, Pittois K, Partridge NC et al. Collagenase-3 (MMP-13) and integral membrane protein 2a (Itm2a) are marker genes of chondrogenic/osteoblastic cells in bone formation: Sequential temporal, and spatial expression of Itm2a, alkaline phosphatase, MMP-13, and osteocalcin in the mouse. J Bone Miner Res 2000;15:1257–1265. 41 Shinmei M, Masuda K, Kikuchi T et al. Production of cytokines by chondrocytes and its role in proteoglycan degradation. J Rheumatol Suppl 1991;27:89 –91. 42 Jime`nez MJ, Balbin M, Lo`pez JM et al. Collagenase 3 is a target of Cbfa1, a transcription factor of the runt gene family involved in bone formation. Mol Cell Biol 1999;19:4431– 4442. 43 Jime`nez MJ, Balbı`n M, Alvarez J et al. A regulatory cascade involving retinoic acid, Cbfa1, and matrix metalloproteinases is coupled to the development of a process of perichondral invasion and osteogenic differentiation during bone formation. J Cell Biol 2001;155:1333–1344. 44 Zhou Z, Apte SS, Soininen R et al. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. Proc Natl Acad Sci U S A 2000;97:4052– 4057. 45 Engsig MT, Chen QJ, Vu TH et al. Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 2000;151:879 – 889. 46 Holmbeck K, Bianco P, Caterina J et al. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell 1999;99:81–92. 47 Murphy G, Knauper V, Cowell S et al. Evaluation of some newer matrix metalloproteinases. Ann N Y Acad Sci 1999;878:25–39. 48 Vu TH, Shipley JM, Bergers G et al. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 1998;93:411– 422. 49 Sakai T, Kambe F, Mitsuyama H et al. Tumor necrosis factor alpha induces expression of genes for matrix degradation in human chondrocyte-like HCS-2/8 cells through activation of NF-kappaB: Abrogation of the tumor necrosis factor alpha effect by proteasome inhibitors. J Bone Miner Res 2001;16:1272–1280. 50 Cancedda R, Descalzi Cancedda F, Castagnola P. Chondrocyte differentiation. Int Rev Cytol 1995;159:265–358. 51 Lee JV, Kim YH, Kim SH et al. Chondrogenic differentiation of mesenchymal stem cells and its clinical applications. Yonsei Med J 2004; 45(suppl):41– 47. 52 Inui T, Ishibashi O, Origane Y et al. Matrix metalloproteinases and lysosomal cysteine proteases in osteoclasts contribute to bone resorption through distinct modes of action. Biochem Biophys Res Commun 1999; 258:173–178.

www.StemCells.com

481

53 Cronwright G, Le Blanc K, Gotherstrom C et al. Cancer/testis antigen expression in human mesenchymal stem cells: Down-regulation of SSX impairs cell migration and matrix metalloproteinase 2 expression. Cancer Res 2005;65:2207–2215. 54 Batouli S, Miura M, Brahim J et al. Comparison of stem-cell-mediated osteogenesis and dentinogenesis. J Dent Res 2003;82:976 –981. 55 Holmbeck K, Bianco P, Pidoux I et al. The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone. J Cell Sci 2005;118:147–156. 56 Karsdal MA, Larsen L, Engsig MT et al. Matrix metalloproteinasedependent activation of latent transforming growth factor-beta controls the conversion of osteoblasts into osteocytes by blocking osteoblast apoptosis. J Biol Chem 2002;277:44061– 44067. 57 Karsdal MA, Andersen TA, Bonewald L et al. Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes: MT1-MMP maintains osteocyte viability. DNA Cell Biol 2004;23:155–165. 58 Holmes SG, Still K, Buttle DJ et al. Chemically modified tetracyclines act through multiple mechanisms directly on osteoclast precursors. Bone 2004;35:471– 478. 59 Cheung R, Erclik MS, Mitchell J. Increased expression of G11alpha in osteoblastic cells enhances parathyroid hormone activation of phospholipase C and AP-1 regulation of matrix metalloproteinase-13 mRNA. J Cell Physiol 2005;204:336 –343. 60 Geoffroy V, Marty-Morieux C, Le Goupil N et al. In vivo inhibition of osteoblastic metalloproteinases leads to increased trabecular bone mass. J Bone Miner Res 2004;19:811– 822. 61 Stamenkovic I. Extracellular matrix remodelling: The role of matrix metalloproteinases. J Pathol 2003;200:448 – 464. 62 Lijnen HR, Maquoi E, Hansen LB et al. Matrix metalloproteinase inhibition impairs adipose tissue development in mice. Arterioscler Thromb Vasc Biol 2002;22:374 –379. 63 Kubo Y, Kaidzu S, Nakajima I et al. Organization of extracellular matrix components during differentiation of adipocytes in long-term culture. In Vitro Cell Dev Biol Anim 2000;36:38 – 44. 64 Brown LM, Fox HL, Hazen SA et al. Role of the matrixin MMP-2 in multicellular organization of adipocytes cultured in basement membrane components. Am J Physiol 1997;272:C937–C949. 65 Johnson MD, Kim HR, Chesler L et al. Inhibition of angiogenesis by tissue inhibitor of metalloproteinase. J Cell Physiol 1994;160:194 –202. 66 Lund LR, Romer J, Thomasset N et al. Two distinct phases of apoptosis in mammary gland involution: Proteinase-independent and -dependent pathways. Development 1996;122:181–193. 67 Alexander CM, Selvarajan S, Mudgett J et al. Stromelysin-1 regulates adipogenesis during mammary gland involution. J Cell Biol 2001;152: 693–703. 68 Bouloumie` A, Sengene`s C, Portolan G et al. Adipocyte produces matrix metalloproteinases 2 and 9: Involvement in adipose differentiation. Diabetes 2001;50:2080:2086. 69 Bourlier V, Zakaroff-Girard A, De Barros S et al. Protease inhibitor treatments reveal specific involvement of matrix metalloproteinase-9 in human adipocyte differentiation. J Pharmacol Exp Ther 2005;312:1272– 1279. 70 Chavey C, Mari B, Monthouel MN et al. Matrix metalloproteinases are differently expressed in adipose tissue during obesity and modulate adipocyte differentiation. J Biol Chem 2003;278:11888 –11896.