Helena Frey

Biological interplay between proteoglycans and their innate immune receptors in inflammation.

An emerging body of evidence indicates that secreted proteoglycans act as signaling molecules, in addition to their canonical function in maintaining and regulating the architecture of various extracellular matrices. Proteoglycans interact with a number of receptors that regulate growth, motility and immune response. In part, as a consequence of their complex structure, proteoglycans can induce crosstalk among various families of receptors and can also interact with natural receptor ligands, often blocking and sequestering their bioactivity. In their soluble form, originating from either partial proteolytic processing or through de novo synthesis by activated cells, some proteoglycans can become potent danger signals, denoting tissue stress and injury. Recently, it has been shown that proteoglycans, especially those belonging to the small leucine‐rich and hyaluronan‐binding gene families as well as the glycosaminoglycan hyaluronan, act as endogenous ligands of the toll‐like receptors, a group of central receptors regulating innate immunity. Furthermore, proteoglycans can activate intracellular inflammasomes and trigger sterile inflammation. In this review, we critically assess the signaling events induced by the proteoglycans biglycan, decorin, lumican and versican as well as hyaluronan during inflammation. We discuss the intriguing emerging notion that, in spite of structural diversity of biglycan, decorin, versican and hyaluronan, all of them signal through the same toll‐like receptors, albeit triggering differential responses and biological outcomes. Finally, we review the modes of action of these endogenous ligands of toll‐like receptors and their ability to specifically modify the final signaling events and the inflammatory response.

Biglycan-triggered TLR-2- and TLR-4-signaling exacerbates the pathophysiology of ischemic acute kidney injury

Exacerbated inflammation in renal ischemia-reperfusion injury, the major cause of intrinsic acute renal failure, is a key trigger of kidney damage. During disease endogenous danger signals stimulate innate immune cells via Toll-like receptors (TLR)-2 and -4 and accelerate inflammatory responses. Here we show that production of soluble biglycan, a small leucine-rich proteoglycan, is induced during reperfusion and that it functions as endogenous agonist of TLR-2/4. Biglycan-mediated activation of TLR-2/4 initiates an inflammatory response in native kidneys, which is marked by the release of cytokines and chemokines and recruitment of inflammatory cells. Overexpression of soluble circulating biglycan before ischemic reperfusion enhanced plasma and renal levels of TNF-α, CXCL1, CCL2 and CCL5, caused influx of neutrophils, macrophages and T cells and overall worsened renal function in wild type mice. We provide robust genetic evidence for TLR-2/4 requirement insofar as biglycan biological effects were markedly dampened in mice deficient in both innate immune receptors, Tlr2(-/-);Tlr4(-/-) mice. Thus, signaling of soluble biglycan via TLR-2/4 could represent a novel therapeutic target for the prevention and possible treatment of patients with acute renal ischemia-reperfusion injury.

Insights into the key roles of proteoglycans in breast cancer biology and translational medicine.

Proteoglycans control numerous normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, vascularization and cancer metastasis. During tumor development and growth, proteoglycan expression is markedly modified in the tumor microenvironment. Altered expression of proteoglycans on tumor and stromal cell membranes affects cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Despite the high complexity and heterogeneity of breast cancer, the rapid evolution in our knowledge that proteoglycans are among the key players in the breast tumor microenvironment suggests their potential as pharmacological targets in this type of cancer. It has been recently suggested that pharmacological treatment may target proteoglycan metabolism, their utilization as targets for immunotherapy or their direct use as therapeutic agents. The diversity inherent in the proteoglycans that will be presented herein provides the potential for multiple layers of regulation of breast tumor behavior. This review summarizes recent developments concerning the biology of selected proteoglycans in breast cancer, and presents potential targeted therapeutic approaches based on their novel key roles in breast cancer.

Decorin is an autophagy-inducible proteoglycan and is required for proper in vivo autophagy.

We have recently discovered that soluble extracellular matrix constituents regulate autophagy via an outside-in signaling pathway. Decorin, a secreted proteoglycan, evokes autophagy in endothelial cells and mitophagy in breast carcinoma cells. However, it is not known whether decorin expression can be regulated by autophagic stimuli such as mTOR inhibition or nutrient deprivation. Thus, we tested whether pro-autophagic stimuli could affect decorin expression in mouse cardiac tissue and whether the absence of decorin could disrupt the in vivo autophagic response. We found that nutrient deprivation induced decorin at the mRNA and protein level in vivo and in vitro, a process regulated at the transcriptional level by inhibiting the canonical mTOR pathway. Moreover, Dcn-/- mice displayed an aberrant response to fasting compared to wild-type mice. Our study establishes a new role for an extracellular matrix proteoglycan and provides a mechanistic role for soluble decorin in regulating a fundamental intracellular catabolic process.

Bimodal role of NADPH oxidases in the regulation of biglycan-triggered IL-1β synthesis

Biglycan, a ubiquitous proteoglycan, acts as a danger signal when released from the extracellular matrix. As such, biglycan triggers the synthesis and maturation of interleukin-1β (IL-1β) in a Toll-like receptor (TLR) 2-, TLR4-, and reactive oxygen species (ROS)-dependent manner. Here, we discovered that biglycan autonomously regulates the balance in IL-1β production in vitro and in vivo by modulating expression, activity and stability of NADPH oxidase (NOX) 1, 2 and 4 enzymes via different TLR pathways. In primary murine macrophages, biglycan triggered NOX1/4-mediated ROS generation, thereby enhancing IL-1β expression. Surprisingly, biglycan inhibited IL-1β due to enhancement of NOX2 synthesis and activation, by selectively interacting with TLR4. Synthesis of NOX2 was mediated by adaptor molecule Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF). Via myeloid differentiation primary response protein (MyD88) as well as Rac1 activation and Erk phosphorylation, biglycan triggered translocation of the cytosolic NOX2 subunit p47(phox) to the plasma membrane, an obligatory step for NOX2 activation. In contrast, by engaging TLR2, soluble biglycan stimulated the expression of heat shock protein (HSP) 70, which bound to NOX2, and consequently impaired the inhibitory function of NOX2 on IL-1β expression. Notably, a genetic background lacking biglycan reduced HSP70 expression, rescued the enhanced renal IL-1β production and improved kidney function of Nox2(-/y) mice in a model of renal ischemia reperfusion injury. Here, we provide a novel mechanism where the danger molecule biglycan influences NOX2 synthesis and activation via different TLR pathways, thereby regulating inflammation severity. Thus, selective inhibition of biglycan-TLR2 or biglycan-TLR4 signaling could be a novel therapeutic approach in ROS-mediated inflammatory diseases.

Heparan Sulfate Proteoglycans Mediate Factor XIIa Binding to the Cell Surface

Background: Factor XIIa (FXIIa) binds to the cell surface; however, the underlying mechanism remains underexplored. Results: Heparan sulfate (HS) enhances FXIIa binding capacity and consequently migration of human lung fibroblasts (HLF) isolated from fibrotic lungs. Conclusion: HS is responsible for local accumulation of FXIIa on the cell surface. Significance: Enhanced association of FXIIa with HLF derived from diseased lungs suggests its role in fibrogenesis. Hageman factor (FXIIa) initiates the intrinsic coagulation pathway and triggers the kallikrein-kinin and the complement systems. In addition, it functions as a growth factor by expressing promitogenic activities toward several cell types. FXIIa binds to the cell surface via a number of structurally unrelated surface receptors; however, the underlying mechanisms are not yet fully understood. Here, we demonstrate that FXIIa utilizes cell membrane-bound glycosaminoglycans to interact with the cell surface of human lung fibroblasts (HLF). The combination of enzymatic, inhibitory, and overexpression approaches identified a heparan sulfate (HS) component of proteoglycans as an important determinant of the FXIIa binding capacity of HLF. Moreover, cell-free assays and competition experiments revealed preferential binding of FXIIa to HS and heparin over dextran sulfate, dermatan sulfate, and chondroitin sulfate A and C. Finally, we demonstrate that fibroblasts isolated from the lungs of the patients suffering from idiopathic pulmonary fibrosis (IPF) exhibit enhanced FXIIa binding capacity. Increased sulfation of HS resulting from elevated HS 6-O-sulfotransferase-1 expression in IPF HLF accounted, in part, for this phenomenon. Application of RNA interference technology and inhibitors of intracellular sulfation revealed the cooperative action of cell surface-associated HS and urokinase-type plasminogen activator receptor in the accumulation of FXIIa on the cell surface of IPF HLF. Moreover, FXIIa stimulated IPF HLF migration, which was abrogated by pretreatment of cells with heparinase I. Collectively, our study uncovers a novel role of HS-type glycosaminoglycans in a local accumulation of FXIIa on the cell membrane. The enhanced association of FXIIa with IPF HLF suggests its contribution to fibrogenesis.

A novel biological function of soluble biglycan: Induction of erythropoietin production and polycythemia

Secondary polycythemia, a disease characterized by a selective increase in circulating mature erythrocytes, is caused by enhanced erythropoietin (Epo) concentrations triggered by hypoxia-inducible factor-2α (HIF-2α). While mechanisms of hypoxia-dependent stabilization of HIF-2α protein are well established, data regarding oxygen-independent regulation of HIF-2α are sparse. In this study, we generated a novel transgenic mouse model, in which biglycan was constitutively overexpressed and secreted by hepatocytes (BGNTg), thereby providing a constant source of biglycan released into the blood stream. We discovered that although the mice were apparently normal, they harbored an increase in mature circulating erythrocytes. In addition to erythrocytosis, the BGNTg mice showed elevated hemoglobin concentrations, hematocrit values and enhanced total iron binding capacity, revealing a clinical picture of polycythemia. In BGNTg mice markedly enhanced Epo mRNA expression was observed in the liver and kidney, while elevated Epo protein levels were found in liver, kidney and blood. Mechanistically, we showed that the transgenic animals had an abundance of HIF-2α protein in the liver and kidney. Finally, by transiently overexpressing circulating biglycan in mice deficient in various Toll-like receptors (TLRs), we determined that this novel function of biglycan to promote Epo synthesis was specifically mediated by a selective interaction with TLR2. Thus, we discovered a novel biological pathway of soluble biglycan inducing HIF-2α protein stabilization and Epo production presumably in an oxygen-independent manner, ultimately giving rise to secondary polycythemia.

Antihistone Properties of C1 Esterase Inhibitor Protect against Lung Injury

&NA; Rationale: Acute respiratory distress syndrome is characterized by alveolar epithelial cell injury, edema formation, and intraalveolar contact phase activation. Objectives: To explore whether C1 esterase inhibitor (C1INH), an endogenous inhibitor of the contact phase, may protect from lung injury in vivo and to decipher the possible underlying mechanisms mediating protection. Methods: The ability of C1INH to control the inflammatory processes was studied in vitro and in vivo. Measurements and Main Results: Here, we demonstrate that application of C1INH alleviates bleomycin‐induced lung injury via direct interaction with extracellular histones. In vitro, C1INH was found to bind all histone types. Interaction with histones was independent of its protease inhibitory activity, as demonstrated by the use of reactive‐center‐cleaved C1INH, but dependent on its glycosylation status. C1INH sialylated‐N‐ and ‐O‐glycans were not only essential for its interaction with histones but also to protect against histone‐induced cell death. In vivo, histone‐C1INH complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury. Furthermore, reactive‐center‐cleaved C1INH attenuated pulmonary damage evoked by intravenous histone instillation. Conclusions: Collectively, C1INH administration provides a new therapeutic option for disorders associated with histone release.

Sphingosine Kinase-2 Deficiency Ameliorates Kidney Fibrosis by Up-Regulating Smad7 in a Mouse Model of Unilateral Ureteral Obstruction

Kidney fibrosis is a hallmark of chronic kidney disease and leads to extracellular matrix accumulation, organ scarring, and loss of kidney function. In this study, we investigated the role of sphingosine kinase-2 (SPHK2) on the progression of tubular fibrosis by using a mouse unilateral ureteral obstruction (UUO) model. We found that SPHK2 protein and activity are up-regulated in fibrotic renal tissue. Functionally, Sphk2-deficient (Sphk2-/-) mice showed an attenuated fibrotic response to UUO compared with wild-type mice, as demonstrated by reduced collagen abundance and decreased expression of fibronectin-1, collagen I, α-smooth muscle actin, connective tissue growth factor (CTGF), and plasminogen activator inhibitor (PAI-1). More important, these changes were associated with increased expression of the antifibrotic protein Smad7 and higher levels of sphingosine in Sphk2-/- UUO kidneys. Mechanistically, sphingosine ameliorates transforming growth factor-β-induced collagen accumulation, CTGF, and PAI-1 expression, but enhances Smad7 protein expression in primary kidney fibroblasts. In a complementary approach, in human Sphk2-overexpressing mice, UUO resulted in exacerbated signs of fibrosis with increased collagen accumulation, higher expression levels of fibronectin-1, collagen I, α-smooth muscle actin, CTGF, and PAI-1, but decreased Smad7 expression. SPHK2 plays an important role in kidney fibrogenesis by modulating transforming growth factor-β signaling. Thus, SPHK2 might be an attractive new target for the treatment of fibrosis in chronic kidney disease.

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.