Andrea Dorfleutner

Regulation of angiogenesis by tissue factor cytoplasmic domain signaling

Hemostasis initiates angiogenesis-dependent wound healing, and thrombosis is frequently associated with advanced cancer. Although activation of coagulation generates potent regulators of angiogenesis, little is known about how this pathway supports angiogenesis in vivo. Here we show that the tissue factor (TF)-VIIa protease complex, independent of triggering coagulation, can promote tumor and developmental angiogenesis through protease-activated receptor-2 (PAR-2) signaling. In this context, the TF cytoplasmic domain negatively regulates PAR-2 signaling. Mice from which the TF cytoplasmic domain has been deleted (TFΔCT mice) show enhanced PAR-2-dependent angiogenesis, in synergy with platelet-derived growth factor BB (PDGF-BB). Ocular tissue from diabetic patients shows PAR-2 colocalization with phosphorylated TF specifically on neovasculature, suggesting that phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of PAR-2 signaling in angiogenesis. Targeting the TF-VIIa signaling pathway may thus enhance the efficacy of angiostatic treatments for cancer and neovascular eye diseases.

Specificity of coagulation factor signaling

Summary.  Coagulation serine proteases signal through protease‐activated receptors (PARs). Thrombin‐dependent PAR signaling on platelets is essential for the hemostatic response and vascular thrombosis, but regulation of inflammation by PAR signaling is now recognized as an important aspect of the pro‐ and anti‐coagulant pathways. In tissue factor (TF)‐dependent initiation of coagulation, factor (F) Xa is the PAR‐1 or PAR‐2‐activating protease when associated with the transient TF–FVIIa–FXa complex. In the anticoagulant protein C (PC) pathway, the thrombin–thrombomodulin complex activates PC bound to the endothelial cell PC receptor (EPCR), which functions as a required coreceptor for activated PC‐mediated signaling through endothelial cell PAR‐1. Thus, the pro‐ and anti‐inflammatory receptor cascades are mechanistically coupled to immediate cell signaling, which precedes systemic coagulant or anticoagulant effects. In contrast to the substrate‐like recognition of PARs by thrombin, TF‐ or EPCR‐targeted activation of PARs generates cell‐type specificity, PAR selectivity and protease receptor cosignaling with the G‐protein‐coupled PAR response. Protease receptors are thus major determinants of the biological outcome of coagulation factor signaling on vascular cells.

An NLRP7-Containing Inflammasome Mediates Recognition of Microbial Lipopeptides in Human Macrophages

Cytosolic pathogen- and damage-associated molecular patterns are sensed by pattern recognition receptors, including members of the nucleotide-binding domain and leucine-rich repeat-containing gene family (NLR), which cause inflammasome assembly and caspase-1 activation to promote maturation and release of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and induction of pyroptosis. However, the contribution of most of the NLRs to innate immunity, host defense, and inflammasome activation and their specific agonists are still unknown. Here we describe identification and characterization of an NLRP7 inflammasome in human macrophages, which is induced in response to microbial acylated lipopeptides. Activation of NLRP7 promoted ASC-dependent caspase-1 activation, IL-1β and IL-18 maturation, and restriction of intracellular bacterial replication, but not caspase-1-independent secretion of the proinflammatory cytokines IL-6 and tumor necrosis factor-α. Our study therefore increases our currently limited understanding of NLR activation, inflammasome assembly, and maturation of IL-1β and IL-18 in human macrophages.

Apoptosis-Associated Speck-Like Protein Containing a Caspase Recruitment Domain Is a Regulator of Procaspase-1 Activation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1β activation by this protease in THP-1 monocytes. ASC enhances IL-1β secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1β secretion but that coexpression of ASC with these proteins results in enhanced IL-1β secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1β secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.

The PAAD/PYRIN-Family Protein ASC Is a Dual Regulator of a Conserved Step in Nuclear Factor κB Activation Pathways

Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk). Recent data have suggested that ASC functions as an adaptor protein linking various PAAD-family proteins to pathways involved in nuclear factor (NF)-κB and pro-Caspase-1 activation. We present evidence here that the role of ASC in modulating NF-κB activation pathways is much broader than previously suspected, as it can either inhibit or activate NF-κB, depending on cellular context. While coexpression of ASC with certain PAAD-family proteins such as Pyrin and Cryopyrin increases NF-κB activity, ASC has an inhibitory influence on NF-κB activation by various proinflammatory stimuli, including tumor necrosis factor (TNF)α, interleukin 1β, and lipopolysaccharide (LPS). Elevations in ASC protein levels or of the PAAD domain of ASC suppressed activation of IκB kinases in cells exposed to pro-inflammatory stimuli. Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IκBα. Our findings suggest that ASC modulates diverse NF-κB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

Activation of Inflammasomes Requires Intracellular Redistribution of the Apoptotic Speck-Like Protein Containing a Caspase Recruitment Domain

Activation of caspase 1 is essential for the maturation and release of IL-1β and IL-18 and occurs in multiprotein complexes, referred to as inflammasomes. The apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is the essential adaptor protein for recruiting pro-caspase 1 into inflammasomes, and consistently gene ablation of ASC abolishes caspase 1 activation and secretion of IL-1β and IL-18. However, distribution of endogenous ASC has not yet been examined in detail. In the present study, we demonstrated that ASC localized primarily to the nucleus in resting human monocytes/macrophages. Upon pathogen infection, ASC rapidly redistributed to the cytosol, followed by assembly of perinuclear aggregates, containing several inflammasome components, including caspase 1 and Nod-like receptors. Prevention of ASC cytosolic redistribution completely abolished pathogen-induced inflammasome activity, which affirmed that cytosolic localization of ASC is essential for inflammasome function. Thus, our study characterized a novel mechanism of inflammasome regulation in host defense.

IL-17 induces monocyte migration in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease which is in part mediated by the migration of monocytes from blood to RA synovial tissue, where they differentiate into macrophages and secrete inflammatory cytokines and chemokines. The T cell cytokine IL-17 is expressed in the RA synovial tissue and synovial fluid. To better understand the mechanism by which IL-17 might promote inflammation, its role in monocyte trafficking was examined. In vivo, IL-17 mediates monocyte migration into sponges implanted into SCID mice. In vitro, IL-17 was chemotactic, not chemokinetic, for monocytes at the concentrations detected in the RA synovial fluid. Further, IL-17-induced monocyte migration was mediated by ligation to IL-17RA and RC expressed on monocytes and was mediated through p38MAPK signaling. Finally, neutralization of IL-17 in RA synovial fluid or its receptors on monocytes significantly reduced monocyte migration mediated by RA synovial fluid. These observations suggest that IL-17 may be important in recruiting monocytes into the joints of patients with RA, supporting IL-17 as a therapeutic target in RA.

COPs and POPs: Modulators of Inflammasome Activity

Inflammasomes represent molecular platforms for the activation of inflammatory caspases and are essential for processing and secretion of the inflammatory cytokines IL-1β and IL-18. Multiple key proteins of inflammasomes contain caspase recruitment domains (CARDs) or pyrin domains (PYDs). Dissecting CARD- and PYD-mediated interactions substantially improved our understanding of the mechanisms by which these protein platforms are activated and emphasized their essential role during the inflammatory cytokine response. However, their precise regulation is still poorly understood. A family of small proteins that are composed of either a CARD or a PYD only emerged as important inflammasome regulators. These CARD-only proteins (COPs) and PYD-only proteins (POPs) function as endogenous dominant negative proteins that modulate the activity of inflammasomes in response to pathogen infection and tissue destruction. In this review we will summarize the most recent advances in the regulation of inflammasomes and highlight their importance for immunity and inflammatory disease.

Caspase-8 promotes cell motility and calpain activity under nonapoptotic conditions.

Significant caspase-8 activity has been found in normal and certain tumor cells, suggesting that caspase-8 possesses an alternative, nonapoptotic function that may contribute to tumor progression. In this article, we report that caspase-8 promotes cell motility. In particular, caspase-8 is required for the optimal activation of calpains, Rac, and lamellipodial assembly. This represents a novel nonapoptotic function of caspase-8 acting at the intersection of the caspase-8 and calpain proteolytic pathways to coordinate cell death versus cell motility signaling.

A Shope Fibroma virus PYRIN-only protein modulates the host immune response

PYRIN domain (PYD) proteins have recently emerged as important signaling molecules involved in the development of innate immunity to intracellular pathogens through activation of inflammatory mediator pathways. ASC is the central adaptor protein, which links pathogen recognition by PYD-containing pathogen recognition receptors to the activation of downstream effectors, including activation of Caspase-1 and NF-κB. The cellular PYD-only protein 1 (cPOP1) can block the recruitment of ASC to activated PAN receptors and thereby functions as an endogenous inhibitor of the PYD-mediated signal transduction pathway. Here we describe the identification and characterization of a Shope Fibroma homolog to cPOP1. Like cPOP1, a Shope Fibroma virus-encoded POP (vPOP), co-localizes and directly associates with ASC and inhibits PYD-mediated signal transduction. Poxviruses are known to encode immune evasive proteins to promote host cell infection and suppression of the host immune response. Poxvirus-encoded vPOPs represent a novel class of immune evasive proteins and impair the host response by blocking Cryopyrin and ASC inflammasome-mediated activation of pro-Caspase-1 and subsequent processing of pro-interleukin (IL)-1β, and expression of vPOPs causes activation of NF-κB.