Growth stimulation of human epidermal cells by urokinase is restricted to the intact active enzyme

Abstract

In this study we report on the effect of urokinase fragments on the proliferation of cells of the human epidermal cell line, CCL 20.2, which expresses high-affinity receptors for urokinase and the growth of which is stimulated by intact active 54-kDa urokinase. The 33-kDa fragment containing only the active-site domain, does not bind to the receptors and does not stimulate cell proliferation, while the 17-kDa fragment, containing only the kringle and the growth-factor domains binds to the receptor but does not stimulate growth of the human epidermal cell line. Growth promotion of this tumor cell line by urokinase is therefore restricted to the complete intact and active urokinase molecule.Activation of plasminogen to plasmin by plasminogen activators may play an important role in the invasion and formation of metastases of tumor cells [l -31. Plasmin is a degradative enzyme capable of digesting the extracellular matrix; it exerts its action either directly on glycoproteins, such as fibronectin or laminin [4,5], or indirectly by activation of latent collagenase [6]. A correlation between cancer and increased plasminogen-activator activity has been found by several groups [3, 71. Significantly elevated levels of plasminogen activator activity in extracts of primary tumors and metastases were found when compared to their normal counterparts [3, 8, 91. Immunological characterization has revealed that most malignant cells produce urokinase-type plasminogen activator (uPA) rather than tissue-type plasminogen activator (tPA) [3, 8, 91. While these data present only indirect support for the involvement of plasminogen activators in malignant processes, more direct evidence can be obtained from the following experiments. Inhibition of plasmin action by tranexamic acid reduced tumor growth of several tumor cell lines implanted into nude mice and inhibited the formation of metastases in rabbits [lo, 111. Moreover, antibodies against urokinase could be shown to inhibit the formation of metastases by a human tumor cell line in an animal model [ 121. We have found previously [ 131 that a human epidermal cell line CCL 20.2, neither containing nor secreting plasminogen activators or plasminogen-activator inhibitors, expresses high-affinity urokinase receptors, similar to those found for several other cell lines [14-161 and that active high-molecular-mass urokinase stimulates proliferation of this cell line. Mitogenic effects of fluid-phase uPA have been previously reported, however, without characterization of urokinase determinants for the observed effects [17]. To prove further that both an intact active site as well as binding of the urokinase molecule to the urokinase receptor are necessary for growth stimulation of the human epidermal tumor cell line, CCL 20.2, we compared, in the present study, the proliferative effect of intact urokinase to that of urokinase fragments. MATERIALS AND METHODS MaterialsTissue-culture cluster plates, 24 flat-bottom wells (Costar, Cambridge, MA); Tween 20, diisopropylfluorophosphate,

Plasminogen activator inhibitor type 2: a regulator of monocyte proliferation and differentiation

“…42 The means by which u-PA/ uPAR transmits such a signal has been the subject of much research. The uPAR-dependent signaling involves the physical engagement with a number of integrins, including the ␤1 and ␤2 integrins and ␣v␤5, and matrix proteins, including vitronectin.…”

Induction of Plasminogen Activator Inhibitor-1 by Urokinase in Lung Epithelial Cells

“…Overexpression of uPA, uPAR, or PAI-1 by tumor cells (31)(32)(33)(34) as well as autoregulatory feedback in normal epithelial cells (35,36) combined with the finding that uPA stimulates proliferation of varied cell types (37)(38)(39)(40)(41)(42) all suggested to us the hypothesis that uPA might regulate PAI-1 expression in lung epithelial cells. Also, the mechanism by which uPA mediates transcellular signaling remains unclear.…”

Design of Novel and Selective Inhibitors of Urokinase-type Plasminogen Activator with Improved Pharmacokinetic Properties for Use as Antimetastatic Agents

“…In a human epidermal tumor cell line CCL20.2 (49) and GUBSB melanoma cells (50), uPA-induced cell proliferation requires uPAR binding and enzymatic activity of uPA. Similar effects were also demonstrated with non-malignant vascular smooth muscle cells, in which only enzymatically intact uPA could induce a mitogenic response (51).…”