USP22 controls necroptosis by regulating receptor-interacting protein kinase 3 ubiquitination.

Dynamic control of ubiquitination by deubiquitinating enzymes is essential for almost all biological processes. Ubiquitin-specific peptidase 22 (USP22) is part of the SAGA complex and catalyzes the removal of mono-ubiquitination from histones H2A and H2B, thereby regulating gene transcription. However, novel roles for USP22 have emerged recently, such as tumor development and cell death. Apart from apoptosis, the relevance of USP22 in other programmed cell death pathways still remains unclear. Here, we describe a novel role for USP22 in controlling necroptotic cell death in human tumor cell lines. Loss of USP22 expression significantly delays TNFα/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFα-mediated NF-κB activation or extrinsic apoptosis. Ubiquitin remnant profiling identified receptor-interacting protein kinase 3 (RIPK3) lysines 42, 351, and 518 as novel, USP22-regulated ubiquitination sites during necroptosis. Importantly, mutation of RIPK3 K518 reduced necroptosis-associated RIPK3 ubiquitination and amplified necrosome formation and necroptotic cell death. In conclusion, we identify a novel role of USP22 in necroptosis and further elucidate the relevance of RIPK3 ubiquitination as crucial regulator of necroptotic cell death.

Affinity Proteomics Identifies Interaction Partners and Defines Novel Insights into the Function of the Adhesion GPCR VLGR1/ADGRV1.

The very large G-protein-coupled receptor 1 (VLGR1/ADGRV1) is the largest member of the adhesion G-protein-coupled receptor (ADGR) family. Mutations in VLGR1/ADGRV1 cause human Usher syndrome (USH), a form of hereditary deaf-blindness, and have been additionally linked to epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying the development of these diseases are still unknown. Our study aimed to identify novel, previously unknown protein networks associated with VLGR1 in order to describe new functional cellular modules of this receptor. Using affinity proteomics, we have identified numerous new potential binding partners and ligands of VLGR1. Tandem affinity purification hits were functionally grouped based on their Gene Ontology terms and associated with functional cellular modules indicative of functions of VLGR1 in transcriptional regulation, splicing, cell cycle regulation, ciliogenesis, cell adhesion, neuronal development, and retinal maintenance. In addition, we validated the identified protein interactions and pathways in vitro and in situ. Our data provided new insights into possible functions of VLGR1, related to the development of USH and epilepsy, and also suggest a possible role in the development of other neuronal diseases such as Alzheimer's disease.

Danger matrix molecules orchestrate CD14/CD44 signaling in cancer development.

The tumor matrix together with inflammation and autophagy are crucial regulators of cancer development. Embedded in the tumor stroma are numerous proteoglycans which, in their soluble form, act as danger-associated molecular patterns (DAMPs). By interacting with innate immune receptors, the Toll-like receptors (TLRs), DAMPs autonomously trigger aseptic inflammation and can regulate autophagy. Biglycan, a known danger proteoglycan, can regulate the cross-talk between inflammation and autophagy by evoking a switch between pro-inflammatory CD14 and pro-autophagic CD44 co-receptors for TLRs. Thus, these novel mechanistic insights provide some explanation for the plethora of reports indicating that the same matrix-derived DAMP acts either as a promoter or suppressor of tumor growth. In this review we will summarize and critically discuss the role of the matrix-derived DAMPs biglycan, hyaluronan, and versican in regulating the TLR-, CD14- and CD44-signaling dialogue between inflammation and autophagy with particular emphasis on cancer development.

Biglycan evokes autophagy in macrophages via a novel CD44/Toll-like receptor 4 signaling axis in ischemia/reperfusion injury.

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44-/- mice but not Cd14-/- mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants.

In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif) ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs) and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1) in a TLR4- and Toll/interleukin (IL)-1R domain-containing adaptor inducing interferon (IFN)-ß (TRIF)-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk)1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions.

Breaking down chronic inflammatory diseases: the role of biglycan in promoting a switch between inflammation and autophagy.

It is well established that biglycan, a small leucine-rich proteoglycan, acts as an extracellular matrix-derived danger signal in its soluble form. By binding to innate immunity Toll-like receptors (TLR) 2 and 4, biglycan initiates and perpetuates the inflammatory response. Previous work has conveyed that biglycan's role in inflammation extends far beyond its function as a canonical danger signal. It has been shown that biglycan acts in an anti-inflammatory capacity, wherein it tightly regulates the inflammatory response. In this review, we will discuss a paradigm shift to our understanding of biglycan signaling in inflammation. Mounting evidence suggests that the selective interactions between biglycan, TLRs, and their adapter proteins critically regulate downstream signaling and disease outcome. Biglycan can act as a high-affinity ligand for TLR coreceptors CD14 and CD44, further providing an additional layer of complexity. We propose a novel concept, that biglycan steers signaling toward inflammation by interacting with CD14, whereas it can trigger autophagy by binding to CD44. Thus, biglycan, and perhaps others soluble proteoglycans, could function as molecular switches which could either propagate the signaling of chronic inflammation or promote the resolution of inflammatory processes. Obviously, these new functions have broad implications in the regulation of various inflammatory diseases and could provide the basis for developing novel therapeutic regimens that would selectively target the interactions between biglycan, TLRs, coreceptors, and adapter molecules.

Biglycan is a new high-affinity ligand for CD14 in macrophages.

Sterile inflammation is a therapeutic target in many diseases where it represents an important initiator of disease progression. However, the detailed mechanisms underlying its evolution and biological relevance are not yet completely elucidated. Biglycan, a prototype extracellular matrix-derived damage-associated molecular pattern, mediates sterile inflammation in macrophages through Toll-like receptor (TLR) 2 and/or TLR4-dependent signaling pathways. Here we discovered that soluble biglycan is a novel high-affinity ligand for CD14, a well-known GPI-anchored co-receptor for TLRs. CD14 is required for all biglycan-mediated TLR2/4 dependent inflammatory signaling pathways in macrophages. By binding to CD14 and choosing different TLR signaling branches, biglycan induced TNF-α and CCL2 via TLR2/4, HSP70 through TLR2, and CCL5 via TLR4. Mechanistically, biglycan evoked phosphorylation and subsequent nuclear translocation of p38, p44/42, and NF-κB, and these effects were due to a specific, high-affinity interaction between biglycan protein core and CD14. Finally, we provide proof-of-principle for the requirement of CD14, by transiently overexpressing biglycan in a mouse model of renal ischemia/reperfusion injury performed in Cd14-/- mice. Lack of Cd14 prevented biglycan-mediated cytokine expression, recruitment of macrophages, M1 macrophage polarization as well as mitigated the tubular damage and serum creatinine levels, thereby improving renal function. Thus, CD14 inhibition could lead to the reduction in the activation of biglycan-TLR2/4 signaling pathways and could be a novel therapeutic approach in inflammatory kidney diseases.

Small Leucine-Rich Proteoglycans in Renal Inflammation: Two Sides of the Coin.

It is now well-established that members of the small leucine-rich proteoglycan (SLRP) family act in their soluble form, released proteolytically from the extracellular matrix (ECM), as danger-associated molecular patterns (DAMPs). By interacting with Toll-like receptors (TLRs) and the inflammasome, the two SLRPs, biglycan and decorin, autonomously trigger sterile inflammation. Recent data indicate that these SLRPs, besides their conventional role as pro-inflammatory DAMPs, additionally trigger anti-inflammatory signaling pathways to tightly control inflammation. This is brought about by selective employment of TLRs, their co-receptors, various adaptor molecules, and through crosstalk between SLRP-, reactive oxygen species (ROS)-, and sphingolipid-signaling. In this review, the complexity of SLRP signaling in immune and kidney resident cells and its relevance for renal inflammation is discussed. We propose that the dichotomy in SLRP signaling (pro- and anti-inflammatory) allows for fine-tuning the inflammatory response, which is decisive for the outcome of inflammatory kidney diseases.