Cynthia E. Wilkins-Port

PAI-1: An Integrator of Cell Signaling and Migration

Cellular migration, over simple surfaces or through complex stromal barriers, requires coordination between detachment/re-adhesion cycles, involving structural components of the extracellular matrix and their surface-binding elements (integrins), and the precise regulation of the pericellular proteolytic microenvironment. It is now apparent that several proteases and protease inhibitors, most notably urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1), also interact with several cell surface receptors transducing intracellular signals that significantly affect both motile and proliferative programs. These events appear distinct from the original function of uPA/PAI-1 as modulators of the plasmin-based proteolytic cascade. The multifaceted interactions of PAI-1 with specific matrix components (i.e., vitronectin), the low-density lipoprotein receptor-related protein-1 (LRP1), and the uPA/uPA receptor complex have dramatic consequences on the migratory phenotype and may underlie the pathophysiologic sequalae of PAI-1 deficiency and overexpression. This paper focuses on the increasingly intricate role of PAI-1 as a major mechanistic determinant of the cellular migratory phenotype.

Regulation of Extracellular Matrix Remodeling following Transforming Growth Factor-β1/Epidermal Growth Factor-Stimulated Epithelial-Mesenchymal Transition in Human Premalignant Keratinocytes

During tumor progression, malignant cells exploit critical developmental and tissue remodeling programs, often promoting a plastic phenotype referred to as an epithelial-mesenchymal transition (EMT). Autocrine/paracrine signaling due to tumor microenvironment cytokines, such as members of the transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) families, largely regulates the morphological and invasive phases of the EMT phenotype. Notably, epithelial cell initiation often coincides with a switch in the response of these cells to TGF-β and is concomitant with EGF receptor amplification. Modeling these events, we have observed that premalignant human keratinocytes, HaCaTs, acquire a highly motile and scattered phenotype indicative of EMT following stimulation with TGF-β1 and EGF. TGF-β1 and EGF have been shown to upregulate a number of matrix metalloproteinases (MMP) in epithelial cells, which may in turn play a role in developing metastatic potential in these cells. We have established that an increase in MMP-10 expression occurs following treatment of HaCaT cells with a combination of TGF-β1 and EGF. This increase in MMP-10 expression paralleled the development of a collagenolytic phenotype that was sensitive to components of the plasminogen activation system, including the plasminogen activator inhibitor type-1 (PAI-1). Significantly high levels of MMP-10 have been detected in squamous cell carcinomas of the head and neck, esophagus, oral cavity and skin. Importantly, TGF-β1 in addition to upregulating MMP-10 has been shown to upregulate PAI-1 expression in HaCaT cells. Taken together, these observations suggest that TGF-β1 and EGF play a complex role in modulating proteolytic and transitional events such as EMT that may facilitate the progression of human premalignant epithelial cells toward a more invasive phenotype.

TGF-β1 + EGF-Initiated Invasive Potential in Transformed Human Keratinocytes Is Coupled to a Plasmin/MMP-10/MMP-1–Dependent Collagen Remodeling Axis: Role for PAI-1

The phenotypic switching called epithelial-to-mesenchymal transition is frequently associated with epithelial tumor cell progression from a comparatively benign to an aggressive, invasive malignancy. Coincident with the emergence of such cellular plasticity is an altered response to transforming growth factor-beta (TGF-beta) as well as epidermal growth factor (EGF) receptor amplification. TGF-beta in the tumor microenvironment promotes invasive traits largely through reprogramming gene expression, which paradoxically supports matrix-disruptive as well as stabilizing processes. ras-transformed HaCaT II-4 keratinocytes undergo phenotypic changes typical of epithelial-to-mesenchymal transition, acquire a collagenolytic phenotype, and effectively invade collagen type 1 gels as a consequence of TGF-beta1 + EGF stimulation in a three-dimensional physiologically relevant model system that monitors collagen remodeling. Enhanced collagen degradation was coupled to a significant increase in matrix metalloproteinase (MMP)-10 expression and involved a proteolytic axis composed of plasmin, MMP-10, and MMP-1. Neutralization of any one component in this cascade inhibited collagen gel lysis. Similarly, addition of plasminogen activator inhibitor type 1 (SERPINE1) blocked collagen degradation as well as the conversion of both proMMP-10 and proMMP-1 to their catalytically active forms. This study therefore identifies an important mechanism in TGF-beta1 + EGF-initiated collagen remodeling by transformed human keratinocytes and proposes a crucial upstream role for plasminogen activator inhibitor type 1-dependent regulation in this event.

PAI-1 is a Critical Upstream Regulator of the TGF-β1/EGF-Induced Invasive Phenotype in Mutant p53 Human Cutaneous Squamous Cell Carcinoma

The emergence of highly aggressive subtypes of human cutaneous squamous cell carcinoma (SCC) often reflects increased autocrine/paracrine TGF-β synthesis and epidermal growth factor receptor (EGFR) amplification. Cooperative TGF-β/EGFR signaling promotes cell migration and induces expression of both proteases and protease inhibitors that regulate stromal remodeling resulting in acquisition of an invasive phenotype. TGF-β1+EGF stimulation increases the production of several matrix metalloproteinases (MMPs) in human SCC. Among the most prominent is MMP-10 which is known to be elevated in SCC in situ. Activation of stromal plasminogen appears to be critical in triggering downstream MMP activity. Paradoxically, PAI-1, the major physiological inhibitor of plasmin generation, is also up-regulated under these conditions and is an early event in progression of incipient epidermal SCC. A model is proposed in which TGF-β1+EGF-dependent MMP-10 elevation directs focalized matrix remodeling events that promote epithelial cell plasticity and tissue invasion. Increased PAI-1 expression serves to temporally and spatially modulate plasmin-initiated pericellular proteolysis, further facilitating epithelial invasive potential. Defining the complex signaling mechanisms that maintain this elegant balance is critical to developing potential therapeutics for the treatment of human cutaneous malignancies.

PAI-1 Regulates the Invasive Phenotype in Human Cutaneous Squamous Cell Carcinoma

The emergence of highly aggressive subtypes of human cutaneous squamous cell carcinoma (SCC) often reflects increased autocrine/paracrine TGF-β synthesis and epidermal growth factor receptor (EGFR) amplification. Cooperative TGF-β/EGFR signaling promotes cell migration and induces expression of both proteases and protease inhibitors that regulate stromal remodeling resulting in the acquisition of an invasive phenotype. In one physiologically relevant model of human cutaneous SCC progression, TGF-β1+EGF stimulation increases the production of several matrix metalloproteinases (MMPs), among the most prominent of which is MMP-10—an MMP known to be elevated in SCC in situ. Activation of stromal plasminogen appears to be critical in triggering downstream MMP activity. Paradoxically, PAI-1, the major physiological inhibitor of plasmin generation, is also upregulated under these conditions and is an early event in progression of incipient epidermal SCC. One testable hypothesis proposes that TGF-β1+EGF-dependent MMP-10 elevation directs focalized matrix remodeling events that promote epithelial cell plasticity and tissue invasion. Increased PAI-1 expression serves to temporally and spatially modulate plasmin-initiated pericellular proteolysis, further facilitating epithelial invasive potential. Defining the complex signaling and transcriptional mechanisms that maintain this delicate balance is critical to developing targeted therapeutics for the treatment of human cutaneous malignancies.

Localization of Urokinase Type Plasminogen Activator to Focal Adhesions Requires Ligation of Vitronectin Integrin Receptors

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the αvβT5 integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the αvβT5 integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either αvβT5 or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the βT1 integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of αvβT3, αvβT5, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the αvβT3 or αvβT5 integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell-matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.

Vitronectin's basic domain is a syndecan ligand which functions in trans to regulate vitronectin turnover.

During the process of tissue remodeling, vitronectin (Vn) is deposited in the extracellular matrix where it plays a key role in the regulation of pericellular proteolysis and cell motility. In previous studies we have shown that extracellular levels of vitronectin are controlled by receptor-mediated endocytosis and that this process is dependent upon vitronectin binding to sulfated proteoglycans. We have now identified vitronectins 12 amino acid “basic domain” which is contained within the larger 40 amino acid heparin binding domain, as a syndecan binding site. Recombinant vitronectins representing wild type vitronectin (rVn) and vitronectin with the basic domain deleted (rVnΔ347–358) were prepared in a baculoviral expression system. The rVn as well as a glutathione S-transferase (GST) fusion protein, consisting of vitronectins 40 amino acid heparin binding domain (GST-VnHBD), exhibited dose dependent binding to HT-1080 cell surfaces, which was attenuated following deletion of the basic domain. In addition, GST-VnHBD supported both HT-1080 and dermal fibroblast cell adhesion, which was also dependent upon the basic domain. Similarly, ARH-77 cells transfected with syndecans -1, -2, or -4, but not Glypican-1, adhered to GST-VnHBD coated wells, while adhesion of these same cells was lost following deletion of the basic domain. HT-1080 cells were unable to degrade rVnΔ347–358. Degradation of rVnΔ347–358 was completely recovered in the presence of GST-VnHBD but not in the presence of GST-VnHBDΔ347–358. These results indicate that turnover of soluble vitronectin requires ligation of vitronectins basic domain and that this binding event can work in transto regulate vitronectin degradation.

Complex Regulation of the Pericellular Proteolytic Microenvironment during Tumor Progression and Wound Repair: Functional Interactions between the Serine Protease and Matrix Metalloproteinase Cascades

Spatial and temporal regulation of the pericellular proteolytic environment by local growth factors, such as EGF and TGF-β, initiates a wide repertoire of cellular responses coupled to a plasmin/matrix metalloproteinase (MMP) dependent stromal-remodeling axis. Cell motility and invasion, tumor metastasis, wound healing, and organ fibrosis, for example, represent diverse events controlled by expression of a subset of genes that encode various classes of tissue remodeling proteins. These include members of the serine protease and MMP families that functionally constitute a complex system of interacting protease cascades and titrated by their respective inhibitors. Several structural components of the extracellular matrix are upregulated by TGF-β as are matrix-active proteases (e.g., urokinase (uPA), plasmin, MMP-1, -3, -9, -10, -11, -13, -14). Stringent controls on serine protease/MMP expression and their topographic activity are essential for maintaining tissue homeostasis. Targeting individual elements in this highly interactive network may lead to novel therapeutic approaches for the treatment of cancer, fibrotic diseases, and chronic wounds.

Degradation of distinct forms of multimeric vitronectin by human fibroblasts

The plasma protein vitronectin is thought to be an important regulator of extravascular plasminogen activation. In previous studies we have shown that a disulfide stabilized multimeric form of vitronectin is endocytosed and degraded by fibroblast cells (T.S. Panetti, P.J. McKeown-Longo, J. Biol. Chem. 268 (1993) 11988-11993; P.J. McKeown-Longo, T.S. Panetti, in: K.T. Preissner, S. Rosenblatt, C. Kost, J. Wegerhoff, D.F. Mosher (Eds.), Biology of Vitronectins and their Receptors, Elsevier Science Publishers, Amsterdam, 1993, pp. 111-118). The preparation of multimeric vitronectin used in these earlier studies was in the form of high molecular weight disulfide-bonded aggregates which were stable in sodium dodecyl sulfate (SDS). To address the question of whether vitronectin needed to be in the form of disulfide stabilized multimers in order to be endocytosed, a multimeric vitronectin, which was not disulfide stabilized, was prepared from vitronectin that had been treated with reducing agent and alkylated with iodoacetamide. The resulting protein migrated as a 65/75 kDa protein on SDS gels in the absence of reducing agent, confirming that this form of vitronectin was no longer stabilized into disulfide-bonded aggregates. However, the protein was still multimeric when analyzed by native gels and could be converted to SDS stable multimers by cross-linking agents. This result demonstrated that reduced and alkylated vitronectin aggregates into multimeric forms which are not stable in SDS. Similar to disulfide stabilized multimers, alkylated multimers of vitronectin bound to sulfated proteoglycans in the extracellular matrix and were endocytosed and degraded. Degradation of both forms of vitronectin was inhibited with arginine-glycine-aspartic acid peptides, an anti-alphavbeta5 antibody and heparin. Chloroquine and wortmannin were also able to inhibit degradation of both forms of vitronectin, indicating that both multimeric forms were following the same endocytic and degradative pathway. These results suggest that the organization of vitronectin into a multimeric form which will be recognized for endocytosis does not require disulfide bond stabilization. This study further suggests that recognition of vitronectin for endocytosis is dependent upon its conversion from a monomeric to a multivalent form (C.E. Wilkins-Port, P.J. McKeown-Longo, Mol. Biol. Cell 8:S:64A (1997).

pAI-1: A Multifunctional seRpIn with complex Roles in cell signaling and Migration

Elevated levels of plasminogen activator inhibitor type-1 (PAI-1) often occur in concert with the conversion of non-motile epithelial elements into a more migratory phenotype. While essential during embryonic development, this restructuring process, referred to as epithelial-to-mesenchymal-transition (EMT) is limited in the adult organism, occurring normally during wound repair or more atypically in tumor progression. Cell motility, the focal point of EMT, requires the coordinate regulation of multiple mechanisms which ensure proper communication between cell surface receptors and the extracellular environment. PAI-1, through multifaceted interactions with both extracellular matrix (ECM) and cell surface constituents plays a critical role in modulating many of these events. This review focuses on the complex role of PAI-1 in the cellular motile program.