Degradation of Glycoprotein and Collagenous Components of the Basement Membrane: Studies with Urokinase-Type Plasminogen Activator, α-Thrombin, and Plasmin

Abstract

Penetration of the basement membrane takes place at several stages of tumor invasion and meta stasis and appears to involve a variety of degradative proteolytic enzymes directly or indirectly under the regulation of malignant cells. 1 ' 2 ' 4 We have therefore evaluated the role of the neutral serine proteases: urokinase (plasminogen activator), α-thrombin, and plasmin in the degradation of intact basement membrane (BM) and BM components. 3 ' 5 ' 7 Homogeneously purified enzymes were incubated with isolated components of the BM and with whole amnion BM. The components comprised acidextracted type IV collagen, pepsin fragments of type IV collagen, type V collagen, laminin and fibronectin.The purity of the enzymes was verified by sodium dodecyl sulfate polyacrylamide gel electro phoresis (SDS-PAGE) and inhibitor studies. 3,5,6 Digestion of the BM components was performed at 25°C using matched enzymatic activities. Urokinase failed to degrade laminin significantly, or any of the other components. Under the same native condi tions, however, plasmin and α-thrombin cleaved laminin and fibronectin into multiple, specific frag ments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or type V collagen. α-Thrombin selectively degraded only the 400,000 M r β-chain of laminin, whereas plasmin degraded both chains. Utilization of a 14 C-labeled laminin revealed that degrada tion by the serine proteases was concentration and time dependent. In addition to metastatic tumor cells, macrophages and polymorphonuclear leukocytes contained significant laminin-degrading proteolytic activity. This activity was enhanced by the addition of plasminogen. Type V collagen was cleaved by α-thrombin and plasmin at 35°C but not at temperatures below 33°C. Electron microscopy demonstrated preservation of the lamina densa fol lowing treatment of whole-amnion BM with any of the neutral serine proteases employed. Immunohistologic studies revealed that laminin, but not type IV collagen, was removed from the whole BM by plasmin treatment.The results suggest that these BM components are poor substrates for urokinase-type plasminogen activator, and plasmin alone cannot degrade the whole BM. 3,5,8,9 The conversion of plasminogen to plasmin, generated through the catalytic activity of plasminogen activator appears to play a significant role in the degradation of the noncollagenous com ponents of the BM in tumor invasion. The plas minogen activator-mediated generation of plasmin also appears to play an important role in activating latent type IV collagenase and in enhancing the enzymatic activity of a heparan sulfate-degrading heparanase (endoglycosidase) derived from invasive tumor cells. 1,2

Inhibition of tumor invasion and extracellular matrix degradation by ubenimex (bestatin)

“…These results indicate that bestatin inhibited the conversion of latent forms of collagenase to the active forms, thus suggesting that plasma membrane aminopeptidases may be involved in the activation mechanism of type IV collagenase as well as the other matrix proteinases such as plasmin or stromelysin. Plasmin has long been known to directly degrade basement membrane components [37] and to activate the latent type IV collagenase produced by the tumor cells [52]. Bestatin only slightly inhibited plasmin activity of the tumor cells against Boc-Val-Leu-Lys-MCA, but this slight effect was not significant (Table 4).…”

“…The results obtained using the inhibitors of serine proteases and collagenases indicate that both classes of enzymes are necessary for the tumor cells to invade the basement membrane structures (Tables2 and 3). The plasmin generated from plasminogen by plasminogen activator could directly degrade basement membrane components [37,38] or activate the latent collagenase produced by the tumor cells [39]. We therefore examined the effect of bestatin on the plasmin activity of SN12M cells using two synthetic substrates (Table 4) and, unexpectedly, slightly increased SN12M cell degradation of Boc-Glu-Lys-Lys-MCA.…”

“…The treatment of cells with bestatin resulted in the remarkable disappearance of the 68 kDa enzyme level and slight reduction of the 72 kDa enzyme level. It is well known that collagenases exist in both latent and active forms, and that the latent forms can be activated by treatment with trypsin, mercuric compounds, or plasmin [37,38]. Thus, bestatin appears to inhibit the activation of 72 kDa latent type IV collagenase to the 68 kDa active form, suggesting that, since bestatin potently inhibited the aminopeptidase activity in tumor cells ( Figure 3) and the preincubation of tumor cells with bestatin showed no inhibitory effect on cell-mediated type IV collagenolysis (Table 2), the amino peptidases in tumor cells are, in part, associated with the activation of type IV collagenase.…”

Clinical Relevance of the Urokinase-Type and Tissue-Type Plasminogen Activators and of Their Type 1 Inhibitor in Breast Cancer

“…10,11 This enyzme degrades collagen type IV, which is a major part of the basement membrane. 12,13 This tumor cell surface-associated proteolysis is the prerequisite for the detachment of tumor cells from their surrounding tissue, which then might lead to tumor cell invasion and metastasis. Tumor cells may also synthesize the plasminogen activator inhibitor type 1 (PAI-1), which blocks the enzymatic activity of u-PA and tissue type plasminogen activator (t-PA).…”

Enolase‐1 expression in the stratum corneum is elevated with parakeratosis of atopic dermatitis and disrupts the cellular tight junction barrier in keratinocytes

“…Previous reports suggested that ENO1 acts as a PLG receptor and concentrates PLG in the pericellular region of tumours and activated immune cells [18][19][20][21][22]. PL is a serine protease that degrades extracellular matrix and TJ proteins [28][29][30][31][32]. Based on these observations, we speculated that ENO1 in keratinocytes may act as a PLG receptor and may cause TJ barrier dysfunction by promoting PL-mediated disruption of cell-cell junctions, as observed in tumour and activated immune cells.…”