Protease Nexin-1 Inhibits Plasminogen Activation-induced Apoptosis of Adherent Cells

Abstract

Degradation of adhesive glycoproteins by plasmin is implicated in cell migration. In this study, we further explored the role of plasminogen activation in cell adhesion and survival and show that uncontrolled plasminogen activation at the cell surface may induce cell detachment and apoptosis. We hypothesized that this process could be prevented in adherent cells by expression of protease nexin-1, a potent serpin able to inhibit thrombin, plasmin, and plasminogen activators. Using two-and three-dimensional culture systems, we demonstrate that Chinese hamster ovary fibroblasts constitutively express tissue-type plasminogen activator and efficiently activate exogenously added plasminogen in a specific and saturable manner (K m ‫؍‬ 46 nM). The formation of plasmin results in proteolysis of fibronectin and laminin, which is followed by cell detachment and apoptosis. Protease nexin-1 expressed by transfected cells significantly inhibited the activity of plasmin and tissue-type plasminogen activator via the formation of inhibitory complexes and prevented cell detachment and apoptosis. In conclusion, protease nexin-1 may be an important anti-apoptotic factor for adherent cells. This cell model could be a useful tool to evaluate therapeutic agents such as serpins in vascular pathologies involving pericellular protease-protease inhibitor imbalance.Apoptosis of adherent cells induced by disruption of integrinmediated cell-matrix interactions, referred to as anoikis, has been described in physiological conditions such as the renewal of intestinal epithelial cells and the involution of the mammary gland after lactation (for a review, see Ref. 1). Recent studies have shown that resistance to anoikis may also contribute to the progression of malignancy (2). Therefore, definition of the intracellular events and extracellular mediators involved in pathological anoikis could be helpful to define therapeutic targets. The intracellular pathways involved in anoikis have been extensively studied and include Bcl-2-related proteins (3), p53 (4), interleukin-1␤ converting enzyme-related proteases (including caspase-3), JNK (c-Jun N-terminal kinase) (5), MEKK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase) proteolysis by caspases (6), focal adhesion kinase (7,8), and Akt (9). In contrast, the extracellular mediators that can trigger anoikis are less well characterized. It has been shown, however, that growth factors (such as insulin, insulin-like growth factor-1, and epidermal growth factor) and their receptors cooperate with integrins to trigger different intracellular survival signaling pathways (2). Proteases such as matrix metalloproteinases (10, 11) and leukocyte elastase (12) could also trigger anoikis by breaking down cell-extracellular matrix interactions. Plasmin, the active enzyme generated by plasminogen activation (13), is able to cleave adhesive glycoproteins such as laminin (14, 15) and fibronectin (16) and can interfere with integrin-mediated cell adhesion to adhesive glycoproteins (1...

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle

“…Plasmin activity in FF appears to be regulated by PN-1, as plasmin activity was correlated with PN-1 but not uPA, and uPA activity did not change significantly with follicular health. PN-1 has been suggested to be an anti-apoptotic factor in adherent cells, as it inhibited plasminogen activation-induced anoikis in these cells (Rossignol et al 2004). Atresia in dominant bovine follicles is characterized by apoptosis and detachment of granulosa cells near the antrum (Irving- Rodgers et al 2001), and in sheep there are changes in collagen and fibronectin content of earlyatretic follicles (Huet et al 1998).…”

“…The PA system may be involved in the growth of the dominant follicle, as cellular uPA activity was higher in granulosa cells from small antral follicles compared with those from large follicles, and PN-1 secretion and expression was lower in granulosa cells from small follicles compared with those of large follicles of rodents and cattle (Hägglund et al 1996, Bédard et al 2003, Cao et al 2004. Follicular regression may also involve PA activity, as plasminogen activation markedly decreased attachment of Chinese hamster ovary fibroblasts to ECM components in vitro, resulting in detachment-induced cell death (anoikis) (Rossignol et al 2004). Interestingly, PN-1 inhibited PAinduced anoikis in these cells (Rossignol et al 2004).…”

“…Follicular regression may also involve PA activity, as plasminogen activation markedly decreased attachment of Chinese hamster ovary fibroblasts to ECM components in vitro, resulting in detachment-induced cell death (anoikis) (Rossignol et al 2004). Interestingly, PN-1 inhibited PAinduced anoikis in these cells (Rossignol et al 2004).…”

SERPINE1 (PAI-1) is deposited into keratinocyte migration “trails” and required for optimal monolayer wound repair

“…PAI-1 mRNA/ protein increase rapidly in cultured epithelial cells immediately adjacent to experimentally-created wounds and remain elevated over the course of monolayer "healing" [12,25,29], closely recapitulating PAI-1 spatial/temporal induction in the wounded epidermis [18,34]. Several SERPINS (i.e., PAI-1, protease nexin-1), in fact, modulate the complex integrative process of injury resolution largely through the focalized regulation of plasmin-initiated matrix remodeling, cell-to-substrate adhesion/detachment, migration and apoptosis [3,[7][8][9][10]24,26,35,40]. The effects of PAI-1 on cell migration, however, are not limited to its proteolytic inhibitory activity.…”

Le devenir des cellules SDF

“…Différents médiateurs extracellulaires de l'anoïkis ont été proposés dans les maladies cardiovasculaires où l'apoptose est impliquée, comme la désendothé-lialisation, l'insuffisance cardiaque, la fragilisation de la plaque d'athérome et la disparition des cellules musculaires lisses dans les parois anévrismales [6]. L'excès d'activité protéase joue un rôle majeur dans la rupture des communications entre cellules et matrice péricellulaire ; ces protéases peuvent être d'origine inflammatoire (élastase, cathepsines, chymase, tryptase, granzymes) ou résulter de l'activation de zymogènes (activation du plasminogène, de la prothrombine…) [7][8][9]. Ces phénomènes d'activation tissulaire sont en majeure partie dus à la liaison de ces enzymes à des structures macromolé-culaires (gel de fibrine, membranes cellulaires, substances amyloïdes), liaisons qui les rendent en partie inaccessibles à leurs inhibiteurs naturels circulants.…”