Shravan K. Chintala, Ph.D. and Jasti S. Rao, Ph.D.

Department of Neurosurgery, Box 064, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.

Received 9/28/96; Accepted 10/17/96; On-line 11/01/96

6. ECM TURNOVER AND DEGRADATION

Matrix degradation and turnover are important processes in tissue remodeling during development, wound healing, tumor necrosis, and inflammation. Key components of ECM turnover and regulation are the matrix metalloproteases (MMP) and their inhibitors TIMPs (tissue inhibitors of metalloproteases). A recently characterized metalloprotease family consists of membrane-type matrix metalloproteases (MT-MMP) that activate progelatinase A and a cascade of matrix proteases that in turn degrade extracellular matrix.

6.1. Matrix Metalloproteases

Matrix metalloproteases (MMPs) are a group of zinc-dependent enzymes that degrade ECM molecules, proteoglycans, glycoproteins, and various types of collagens (126, 127). MMPs are secreted by cells in a prometalloprotease form. The activation of MMPs in vivo is poorly understood. The MMP family has nine members that can be divided into three distinct groups based on their substrate specificities (table 2).

Often, the activation of the enzyme is completed by an autocatalytic activation that results in the loss of an aminoterminal peptide (130). MMP may be activated in vitro by detergents, organomercurial compounds, and proteolytic enzymes such as plasmin, trypsin, kallikrein and stromelysin (127-131). All these agents induce a conformational change such that the cystein-residue in the propeptide and the zinc molecule in the enzyme is disrupted exposing the zinc molecule (131). This, in turn, results in a partially active intermediate enzyme that cleaves the propeptide by autocatalytic activation and results in an active enzyme. Local invasive growth is one of the key features of primary brain tumors. The most common and malignant brain tumors, glioblastoma multiforme, is characterized by aggressive invasion of the surrounding normal brain. The specific mechanisms for this invasive behavior is still obscure; however, tumor cell interaction with the immediate ECM and subsequent degradation of the extracellular matrix components plays a key role in this process. Studies have shown that both MMP-2 (132) and MMP-9 (133) were significantly higher in malignant brain tumors in vivo. Moreover, MMP-9 facilitates the invasion of glioblastoma cells in vitro (25) and MMP-9 over expression correlates with the malignant progression of gliomas in vivo (134). MMPs function is regulated by naturally occurring protease inhibitors, tissue inhibitors of metalloproteases (TIMPs). The TIMP family consists of three members, TIMP-1 (a 28-kDa protein), TIMP-2 (a 21-kDa protein), and TIMP-3 (a 21-kDa protein) all with broad specificity in inhibiting MMPs (Table 2). However, the ability of these inhibitors to form complexes with latent and proenzyme forms of various types of collagenases differs significantly. Decreased levels of TIMP-1 and TIMP-2 expression favors the invasion of glioblastoma cells (135).

Membrane-type matrix metalloproteases are, recently discovered, distinct metalloproteases that are expressed on the surface of tumor cells. MT-MMPs activate gelatinase and also function as receptors for gelatinase A (136-138), inducing efficient pericellular proteolysis. So far, MT-MMP-1, MT-MMP-2, and MT-MMP-3 have been identified (139). MT-MMP expression has been found to correlate with the invasive behavior of HT-1080 and NIH3T3 cells (140). Our recent studies showed MT-MMP-1 to be highly upregulated in human glioblastomas (141).

6.2. Plasminogen activators

Plasminogen activators, (PAs) which belong to the serine protease family, are involved in matrix degradation. Two types of PA: urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) convert plasminogen to plasmin, a broad-spectrum enzyme that cleaves fibrin, fibronectin, proteoglycans, and laminin (142, 143). uPA is involved in tissue remodeling during wound healing, inflammatory cellular migration, neo-vascularization and tumor cell invasion, while tPA, a key enzyme in thrombosis is involved in the dissolution of clots in blood vessels and the maintenance of hemostasis in the vasculature (144). tPA is expressed during brain development (145) and may be involved in neuronal migration and neurite outgrowth (146, 147). On the other hand, tPA is totally absent in glioblastoma, colon, lung and breast metastasis. However, anaplastic astrocytoma, low grade glioma and meningioma contain normal levels of tPA (148).

uPA is a highly specific serine protease that is secreted as zymogen, as a single chain (sc) polypeptide (scu-PA), and is readily converted into an active two-chain uPA (tcu-PA) by plasmin, kallikrein, cathepsin B, and nerve growth factor-gamma (143, 149-152). Receptor-bound active tcu-PA increases the rate at which plasminogen converts to plasmin (142, 143). The major substrate for uPA is plasmin, which has trypsin-like broad-spectrum protease activity, and which activates MMP (153, 154). uPA is synthesized by various tissues and cells, including those of CNS. One of the plasminogen activator, uPA binds to a specific cell surface receptor, uPAR and provides a localized cell surface proteolysis for the degradation of ECM. Proteolytic activity of uPA is modulated by its cell surface receptor, as well as by plasminogen activator inhibitors (PAIs). Recent studies demonstrated that overexpression of uPA was associated with malignant progression of human gliomas (155) and high affinity binding uPARs significantly contributes to the invasive behavior of gliomas in vitro (156). In situ hybridization studies have shown that uPAR mRNA was significantly higher in glioblastomas than in low grade glioma and normal brain (157).

Plasminogen activators are regulated by plasminogen activator inhibitors (PAIs) and protease nexin-I (PN-1). PAI-1 is a 46-54 kDa glycoprotein with high specificity for both uPA and tPA (158). Recent immunohistochemical and biochemical studies (159) have shown that the amount of PAI-1 was higher in malignant brain tumors. Using northern blot and in situ hybridization techniques, Yamamoto et al. (160) have shown higher levels of PAI-1 mRNA in glioblastoma than normal brain tissue. Recent evidence also indicates a correlation between the presence of tumor necrosis and higher levels of PAI-1 in glioblastomas (161). PAI-2, originally found in placental tissue (162), has a stronger affinity for uPA than tPA; it also exists in plasma. PAI-3, a 50-kDa inhibitor, has stronger affinity for uPA than tPA. The protease nexin-1 is a 43-50 kDa secreted glycoprotein, that inhibits uPA and plasmin. In the ECM, nexin-1 binds to thrombin, urokinase and plasmin (163). PN-1 is present in normal human brain (164), is produced mainly by astrocytes (165) and by glioblastoma cells (166, 167). Higher PN-1 levels have been observed in an experimental rat 9L gliosarcoma tumor than in normal brain (167).