Apical sodium-dependent bile acid transporter inhibition with volixibat improves metabolic aspects and components of non-alcoholic steatohepatitis in Ldlr-/-.Leiden mice.

Interruption of bile acid recirculation through inhibition of the apical sodium-dependent bile acid transporter (ASBT) is a promising strategy to alleviate hepatic cholesterol accumulation in non-alcoholic steatohepatitis (NASH), and improve the metabolic aspects of the disease. Potential disease-attenuating effects of the ASBT inhibitor volixibat (5, 15, and 30 mg/kg) were investigated in high-fat diet (HFD)-fed Ldlr-/-.Leiden mice over 24 weeks. Plasma and fecal bile acid levels, plasma insulin, lipids, and liver enzymes were monitored. Final analyses included liver histology, intrahepatic lipids, mesenteric white adipose tissue mass, and liver gene profiling. Consistent with its mechanism of action, volixibat significantly increased the total amount of bile acid in feces. At the highest dose, volixibat significantly attenuated the HFD-induced increase in hepatocyte hypertrophy, hepatic triglyceride and cholesteryl ester levels, and mesenteric white adipose tissue deposition. Non-alcoholic fatty liver disease activity score (NAS) was significantly lower in volixibat-treated mice than in the HFD controls. Gene profiling showed that volixibat reversed the inhibitory effect of the HFD on metabolic master regulators, including peroxisome proliferator-activated receptor-γ coactivator-1ß, insulin receptor, and sterol regulatory element-binding transcription factor 2. Volixibat may have beneficial effects on physiological and metabolic aspects of NASH pathophysiology.

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-ß, 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-ß 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.

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.

PAI-1 mediates the TGF-beta1+EGF-induced "scatter" response in transformed human keratinocytes.

Cooperative interactions between growth factor signaling pathways are important elements in carcinoma progression. A model system combining transforming growth factor-beta1 (TGF-beta1) and EGF was developed to investigate mechanisms underlying induced epithelial-to-mesenchymal transition (EMT) in ras-transformed human (HaCaT II-4) keratinocytes. Dual stimulation with TGF-beta1+EGF resulted in keratinocyte "plasticity" and pronounced colony dispersal. The most highly expressed transcript, identified by mRNA profiling, encoded plasminogen activator inhibitor-1 (PAI-1; SERPINE1). PAI-1 negatively regulates plasmin-dependent matrix degradation, preserving a stromal scaffold permissive for keratinocyte motility. Mitogen-activated extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) and p38 signaling were required for maximal PAI-1 upregulation and TGF-beta1+EGF-stimulated cell locomotion, as pharmacologic disruption of MEK/p38 activity ablated both responses. Moreover, PAI-1 knockdown alone effectively inhibited TGF-beta1+EGF-dependent cell scattering, indicating a functional role for this SERPIN in the dual-growth factor model of induced motility. Moreover, EGFR signaling blockade or EGFR knockdown attenuated TGF-beta1-induced PAI-1 expression, implicating EGFR transactivation in TGF-beta1-stimulated PAI-1 expression, and reduced colony dispersal in TGF-beta1+EGF-treated cultures. Identification of such cooperative signaling networks and their effect on specific invasion-promoting target genes, such as PAI-1, may lead to the development of pathway-specific therapeutics that affect late-stage events in human tumor progression.

TGF-beta1 + 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.

TGF-ß1-Induced Expression of the Anti-Apoptotic PAI-1 Protein Requires EGFR Signaling.

TGF-ß1 and its target gene encoding plasminogen activator inhibitor-1 (PAI-1) are major regulators of capillary outgrowth, vessel maturation and angiogenic network stability. The increasing realization of the complexity of PAI-1 action in the vascular system requires analysis of specific signaling events that impact its expression in a physiologically-relevant cell system. PAI-1 was required for tubular differentiation and maintenance of cellular survival in complex gels since targeted disruption of PAI-1 synthesis or activity with antisense constructs or function-blocking antibodies resulted in network regression. Indeed, serum-deprivation-induced apoptosis of tubulogenic T2 cells was concentration-dependently inhibited by addition of a stable PAI-1 mutant protein consistent with the established pro-survival role of PAI-1 in vascular endothelial cells. PAI-1 induction and ERK pathway activation in response to TGF-ß1 was attenuated by EGFR signaling blockade (with AG1478) or preincubation with the MMP/ADAM inhibitor GM6001. The combination of AG1478 + GM6001 completely ablated both responses suggesting that EGFR transactivation is important in PAI-1 gene control and may, at least partially, involve ligand shedding. TGF-ß1-stimulated PAI-1 induction was preceded, in fact, by EGFR phosphorylation on Y845 (a src kinase target residue). EGFR1 knockdown with lentiviral shRNA constructs, moreover, effectively decreased (by >75%) TGF-ß1-stimulated PAI-1 expression whereas infection with control (i.e. GFP) viruses had no effect. TGF-ß1 failed to induce PAI-1 synthesis in EGFR-deficient fibroblasts while introduction of a wild-type EGFR1 construct in EGFR(-/-) cells rescued the PAI-1 response to TGF-ß1 confirming, at a genetic level, the targeted knockdown data. The continued clarification of novel cooperative signaling cascades that impact expression of important angiogenic genes (e.g. PAI-1) may provide therapeutically useful targets to manage the pathophysiology of human neoplastic and vascular diseases.

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-beta synthesis and epidermal growth factor receptor (EGFR) amplification. Cooperative TGF-beta/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-beta1+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-beta1+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.

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

Cutaneous tissue injury, both in vivo and in vitro, initiates activation of a "wound repair" transcriptional program. One such highly induced gene encodes plasminogen activator inhibitor type-1 (PAI-1, SERPINE1). PAI-1-GFP, expressed as a fusion protein under inducible control of +800 bp of the wound-activated PAI-1 promoter, prominently "marked" keratinocyte migration trails during the real-time of monolayer scrape-injury repair. Addition of active recombinant PAI-1 to wounded wild-type keratinocyte monolayers as well as to PAI-1(-/-) MEFs and PAI-1(-/-) keratinocytes significantly stimulated directional motility above basal levels in all cell types. PAI-1 expression knockdown or antibody-mediated functional inhibition, in contrast, effectively attenuated injury repair. The defect in wound-associated migratory activity as a consequence of antisense-mediated PAI-1 down-regulation was effectively reversed by addition of recombinant PAI-1 immediately after scrape injury. One possible mechanism underlying the PAI-1-dependent motile response may involve fine control of the keratinocyte substrate detachment/re-attachment process. Exogenous PAI-1 significantly enhanced keratinocyte spread cell "footprint" area while PAI-1 neutralizing antibodies, but not control non-immune IgG, effectively inhibited spreading with apoptotic hallmarks evident within 24 h. Importantly, PAI-1 not only stimulated keratinocyte adhesion and wound-initiated planar migration but also rescued keratinocytes from plasminogen-induced substrate detachment/anoikis. The early transcriptional response of the PAI-1 gene to monolayer trauma and its prominence in the injury repair genetic signature are consistent with its function as both a survival factor and regulator of the time course of epithelial migration as part of the cutaneous injury response program.

Regulation of extracellular matrix remodeling following transforming growth factor-beta1/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-beta (TGF-beta) 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-beta 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-beta1 and EGF. TGF-beta1 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-beta1 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-beta1 in addition to upregulating MMP-10 has been shown to upregulate PAI-1 expression in HaCaT cells. Taken together, these observations suggest that TGF-beta1 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.

PAI-1 is a Critical Upstream Regulator of the TGF-beta1/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-beta synthesis and epidermal growth factor receptor (EGFR) amplification. Cooperative TGF-beta/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-beta1+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-beta1+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.

The uPA receptor and the somatomedin B region of vitronectin direct the localization of uPA to focal adhesions in microvessel endothelial cells.

Vitronectin is a plasma protein which can deposit into the extracellular matrix where it supports integrin and uPA dependent cell migration. In earlier studies, we have shown that the plasma protein, vitronectin, stimulates focal adhesion remodeling by recruiting urokinase-type plasminogen activator (uPA) to focal adhesion sites [Wilcox-Adelman, S. A., Wilkins-Port, C. E., McKeown-Longo, P. J., 2000. Localization of urokinase-type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. Cell. Adhes. Commun.7, 477-490]. In the present study, we used a variety of vitronectin constructs to demonstrate that the localization of uPA to adhesion sites requires the binding of both vitronectin integrin receptors and the uPA receptor (uPAR) to vitronectin. A recombinant fragment of vitronectin containing the connecting sequence (VN(CS)) was able to support integrin-dependent adhesion, spreading and focal adhesion assembly by human microvessel endothelial cells. Cells adherent to this fragment were not able to localize uPA to focal adhesions. A second recombinant fragment containing both the amino-terminal SMB domain and the CS domain was able to restore the localization of uPA to adhesion sites. This fragment, which contains a uPAR binding site, also resulted in the localization of uPAR to adhesion sites. uPAR blocking antibodies as well as phospholipase C treatment of cells inhibited uPA localization to adhesion sites confirming a role for uPAR in this process. The SMB domain alone was unable to direct either uPAR or uPA to adhesion sites in the absence of the CS domain. Our results indicate that vitronectin-dependent localization of uPA to adhesion sites requires the sequential binding of vitronectin integrins and uPAR to vitronectin.

The receptor for urokinase-type plasminogen activator regulates fibronectin matrix assembly in human skin fibroblasts.

Previous studies have indicated that the receptor for urokinase-type plasminogen activator, uPAR, can form functional complexes with integrin receptors thereby modulating integrin activity. In the present study, the role of uPAR in the regulation of alpha5beta1-dependent polymerization of the fibronectin matrix was investigated. Incubation of fibroblast monolayers with the P-25 peptide, a uPAR ligand, resulted in a 12-15-fold increase in the accumulation of exogenous fibronectin in the cell layer. The exogenous fibronectin co-localized in the extracellular matrix with endogenous cell-derived fibronectin, and its deposition into the matrix was inhibited by blocking antibodies against the beta1 integrin receptor. The P-25-dependent increase in fibronectin assembly was associated with a 7-8-fold increase in the expression of matrix assembly sites as well as a 37-fold increase in the rate of transfer of cell surface-bound fibronectin into a detergent-insoluble matrix. The effects of P-25 on the matrix assembly were attenuated by incubating cells with either phospholipase C or with antibodies against uPAR, confirming a role for uPAR in the P-25-dependent increase in matrix assembly. P-25-treated cells exhibited a 10-fold increase in the binding of the 120-kDa cell-binding fragment of fibronectin suggesting an increase in alpha5beta1 affinity for fibronectin. Consistent with this, treatment of cells with P-25 also resulted in a 6-10-fold increase in the binding of two different monoclonal antibodies that recognize the active conformation of the beta1 integrin. These results indicate that P-25 increases matrix assembly by altering the activation state of the alpha5beta1 integrin receptor and suggest that changes in integrin activation affect both the number of matrix assembly sites as well as the rate of transfer of cell-bound fibronectin into a detergent-insoluble matrix. These data provide direct evidence that uPAR and integrin receptors synergistically regulate the levels of fibronectin in the extracellular matrix.

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 vitronectin's 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 (rVnDelta347-358) were prepared in a baculoviral expression system. The rVn as well as a glutathione S-transferase (GST) fusion protein, consisting of vitronectin's 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 rVnDelta347-358. Degradation of rVnDelta347-358 was completely recovered in the presence of GST-VnHBD but not in the presence of GST-VnHBDDelta347-358. These results indicate that turnover of soluble vitronectin requires ligation of vitronectin's basic domain and that this binding event can work in trans to regulate vitronectin degradation.