Tessa M. Simone

Resveratrol Causes COX-2- and p53-Dependent Apoptosis in Head and Neck Squamous Cell Cancer Cells

Cyclooxygenase‐2 (COX‐2) content is increased in many types of tumor cells. We have investigated the mechanism by which resveratrol, a stilbene that is pro‐apoptotic in many tumor cell lines, causes apoptosis in human head and neck squamous cell carcinoma UMSCC‐22B cells by a mechanism involving cellular COX‐2. UMSCC‐22B cells treated with resveratrol for 24 h, with or without selected inhibitors, were examined: (1) for the presence of nuclear activated ERK1/2, p53 and COX‐2, (2) for evidence of apoptosis, and (3) by chromatin immunoprecipitation to demonstrate p53 binding to the p21 promoter. Stilbene‐induced apoptosis was concentration‐dependent, and associated with ERK1/2 activation, serine‐15 p53 phosphorylation and nuclear accumulation of these proteins. These effects were blocked by inhibition of either ERK1/2 or p53 activation. Resveratrol also caused p53 binding to the p21 promoter and increased abundance of COX‐2 protein in UMSCC‐22B cell nuclei. Resveratrol‐induced nuclear COX‐2 accumulation was dependent upon ERK1/2 activation, but not p53 activation. Activation of p53 and p53‐dependent apoptosis were blocked by the COX‐2 inhibitor, NS398, and by transfection of cells with COX‐2‐siRNA. In UMSCC‐22B cells, resveratrol‐induced apoptosis and induction of nuclear COX‐2 accumulation share dependence on the ERK1/2 signal transduction pathway. Resveratrol‐inducible nuclear accumulation of COX‐2 is essential for p53 activation and p53‐dependent apoptosis in these cancer cells. J. Cell. Biochem. 104: 2131–2142, 2008.

Chemical Antagonists of Plasminogen Activator Inhibitor-1: Mechanisms of Action and Therapeutic Potential in Vascular Disease

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) is a member of the serine protease inhibitor (SERPIN) superfamily and the predominant physiologic inhibitor of urokinase (uPA) and tissue-type (tPA) plasmingen activators. This system effectively restricts, both spatially and temporally, the conversion of plasminogen to plasmin, thereby regulating physiologic and pathophysiologic stromal remodeling. Dysregulation of this cascade frequently results in anomalies of the tissue repair response. Elevated PAI-1 levels are a causative factor in various forms of vascular disease and tissue fibrotic syndromes. Independent of its role in proteolysis, PAI-1 stimulates cell motility via interacting with low-density lipoprotein receptor-related protein-1 (LRP1) activating several cellular signaling pathwaays. PAI-1 also regulates the availability of cell-surface integrins by promoting their endocytosis in an LRP-1- dependent manner via PAI-1/uPA/uPAR (uPA receptor)/LRPI/integrin complexes. This process fine tunes the special control of pericellular proteolysis and the overall cadence of cell detachment/re-adhesion required for efficient cell migration. These data suggest that PAI-1 modulates cell motility under several contexts, both via by its established anti-proteolytic properties and as a signaling initiator.

A small molecule PAI-1 functional inhibitor attenuates neointimal hyperplasia and vascular smooth muscle cell survival by promoting PAI-1 cleavage.

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of urokinase-and tissue-type plasminogen activators (uPA and tPA), is an injury-response gene implicated in the development of tissue fibrosis and cardiovascular disease. PAI-1 mRNA and protein levels were elevated in the balloon catheter-injured carotid and in the vascular smooth muscle cell (VSMC)-enriched neointima of ligated arteries. PAI-1/uPA complex formation and PAI-1 antiproteolytic activity can be inhibited, via proteolytic cleavage, by the small molecule antagonist tiplaxtinin which effectively increased the VSMC apoptotic index in vitro and attenuated carotid artery neointimal formation in vivo. In contrast to the active full-length serine protease inhibitor (SERPIN), elastase-cleaved PAI-1 (similar to tiplaxtinin) also promoted VSMC apoptosis in vitro and similarly reduced neointimal formation in vivo. The mechanism through which cleaved PAI-1 (CL-PAI-1) stimulates apoptosis appears to involve the TNF-α family member TWEAK (TNF-α weak inducer of apoptosis) and its cognate receptor, fibroblast growth factor (FGF)-inducible 14 (FN14). CL-PAI-1 sensitizes cells to TWEAK-stimulated apoptosis while full-length PAI-1 did not, presumably due to its ability to down-regulate FN14 in a low density lipoprotein receptor-related protein 1 (LRP1)-dependent mechanism. It appears that prolonged exposure of VSMCs to CL-PAI-1 induces apoptosis by augmenting TWEAK/FN14 pro-apoptotic signaling. This work identifies a critical, anti-stenotic, role for a functionally-inactive (at least with regard to its protease inhibitory function) cleaved SERPIN. Therapies that promote the conversion of full-length to cleaved PAI-1 may have translational implications.

Small Molecule Targeting of PAI-1 Function: A New Therapeutic Approach for Treatment of Vascular Stenosis.

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) is a clade E1 member of the serine protease inhibitor (SERPIN) superfamily and the major physiologic inhibitor of the urokinase (uPA) and tissue-type (tPA) plasminogen activators. Elevated PAI-1 expression is a significant causative factor in vascular disease and a major contributor to the pathophysiology of diabetes, metabolic syndrome, stroke, atherosclerosis and restenosis, particularly in the setting of increased vessel TGF-β1 [1–3]. PAI-1 is unique relative to other SERPINs existing in the structurally and functionally distinct active, latent and cleaved conformations [4, 5]. PAI-1 is initially synthesized in an active state, capable of interacting with its proteinase targets, but is unstable (half-life of 2 hours at 37°C, pH 7.4) and converts spontaneously into a latent form [6]. Latency requires insertion of the N-terminus of the PAI-1 reactive center loop into β-sheet A forming a new β-strand (s4A) which creates an unusual loop structure and conformational change in the reactive center, ultimately preventing interaction with proteinases [7–9]. Alternatively, PAI-1 can be proteolytically-cleaved at the sissile P1-P1’ bond causing the N-terminal end of the reactive center loop to insert into β-sheet A, while the C-terminus of the reactive site loop forms strand s1C in β-sheet C. These structural rearrangments produce a 70A separation of the P1 and P1’ residues, thereby, preventing PAI-1 from complexing with the target proteinase due to spatial distortion, ultimately allowing for increased plasmin activation [10–12]. While neither cleaved nor latent PAI-1 forms complexes with their target proteases, all three conformations bind the low-density lipoprotein receptor-related protein-1 (LRP1) and initiate Jak/Stat signaling [13]. Elevated PAI-1 mRNA and protein expression are evident in the carotid vascular wall adjacent to thrombi induced by implantation of indwelling polyethylene tubing [14]. Furthermore, adenoviral delivery of PAI-1 potentiated neointima formation after catheter-induced injury while copper-stimulated neointima formation was reduced in PAI-1-null mice [15, 16]. In a mouse model of carotid artery ligation, PAI-1 protein levels are elevated in neointimal lesions 14-days after restriction. Regions expressing PAI-1 also express smooth muscle cell α-actin (Figure 1A,C), suggesting that PAI-1 is associated with smooth muscle cells (VSMCs). PAI-1 involvement in the pathological response to healing is reflected in its expression in the developing neointima in the ligated artery, but not the contralateral control vessel (Figure 1B,D), as well as in balloon-injured carotid arteries (Figure 1E). These findings implicate PAI-1 as a significant factor in the development of restenosis and provided the impetus for development of low-molecular weight PAI-1 antagonists. Tiplaxtinin (PAI-039), the most well studied small molecule PAI-1 inhibitor, attenuates asthmatic episodes, obesity, diabetes, cancer cell motility and angiogenesis [17–24]. The mechanism by which Tiplaxtinin antagonizes the anti-fibrinolytic activity of PAI-1 appears to involve inhibition of complex formation between PAI-1 and its target protease with promotion of PAI-1 cleavage [25, 26]. This has translational implications as PAI-1 deficiency in various cell types promotes plasmin-dependent apoptosis [27–31]. A decrease in PAI-1 antiproteolytic activity, through functional blockade or proteolytic cleavage, may subsequently increase VSMC apoptosis due to plasmin generation. One mechanism suggests that PAI-1 might contribute to neointimal growth by facilitating VSMC survival. Recent findings indicate that Tiplaxtinin induces VSMC apoptosis in a dose-dependent manner and this response was attenuated by the addition of TGF-β1. However, with the exception that PAI-1 binds and prevents the cleavage and activation of caspase-3, the role of PAI-1 in preventing VSMC apoptosis remains unexplored [31]. One attractive possibility is that PAI-1 might promote cell survival through the PI3K/Akt signaling axis and both PAI-1 and TGF- β1 stimulate AKT phosphorylation. Since PAI-1 is a highly upregulated gene in the TGF-β1 response set, TGF-β1 may activate Akt through PAI-1 or, at least, induce PAI-1 expression through an Akt-dependent pathway. Given the ubiquitous role PAI-1 plays in the etiology and progression of several chronic and acute fibrotic disorders, the therapeutic efficacy of small molecule PAI-1 inhibitors, such as Tiplaxtinin, may have translational adapatability beyond the scope of vascular disease. Figure 1 PAI-1 expression is upregulated within vascular smooth muscle cells of neointimal lesions

The Basic Helix-Loop-Helix/Leucine Zipper Transcription Factor USF2 Integrates Serum-Induced PAI-1 Expression and Keratinocyte Growth

Plasminogen activator inhibitor type‐1 (PAI‐1), a major regulator of the plasmin‐dependent pericellular proteolytic cascade, is prominently expressed during the tissue response to injury although the factors that impact PAI‐1 induction and their role in the repair process are unclear. Kinetic modeling using established biomarkers of cell cycle transit (c‐MYC; cyclin D1; cyclin A) in synchronized human (HaCaT) keratinocytes, and previous cytometric assessments, indicated that PAI‐1 transcription occurred early after serum‐stimulation of quiescent (G0) cells and prior to G1 entry. It was established previously that differential residence of USF family members (USF1→USF2 switch) at the PE2 region E box (CACGTG) characterized the G0 → G1 transition period and the transcriptional status of the PAI‐1 gene. A consensus PE2 E box motif (5′‐CACGTG‐3′) at nucleotides −566 to −561 was required for USF/E box interactions and serum‐dependent PAI‐1 transcription. Site‐directed CG → AT substitution at the two central nucleotides inhibited formation of USF/probe complexes and PAI‐1 promoter‐driven reporter expression. A dominant‐negative USF (A‐USF) construct or double‐stranded PE2 “decoy” attenuated serum‐ and TGF‐β1‐stimulated PAI‐1 synthesis. Tet‐Off induction of an A‐USF insert reduced both PAI‐1 and PAI‐2 transcripts while increasing the fraction of Ki‐67+ cells. Conversely, overexpression of USF2 or adenoviral‐delivery of a PAI‐1 vector inhibited HaCaT colony expansion indicating that the USF1 → USF2 transition and subsequent PAI‐1 transcription are critical events in the epithelial go‐or‐grow response. Collectively, these data suggest that USF2, and its target gene PAI‐1, regulate serum‐stimulated keratinocyte growth, and likely the cadence of cell cycle progression in replicatively competent cells as part of the injury repair program. J. Cell. Biochem. 115: 1840–1847, 2014.