Ute Raffetseder

Y-Box Binding Protein-1 Controls CC Chemokine Ligand-5 (CCL5) Expression in Smooth Muscle Cells and Contributes to Neointima Formation in Atherosclerosis-Prone Mice

Background— The CC chemokine CCL5/Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) is upregulated in mononuclear cells or deposited by activated platelets during inflammation and has been implicated in atherosclerosis and neointimal hyperplasia. We investigated the influence of the transcriptional regulator Y-box binding protein (YB)-1 on CCL5 expression and wire-induced neointimal hyperplasia. Methods and Results— Analysis of the CCL5 promoter revealed potential binding sites for YB-1, and interaction of YB-1 with a sequence at position −204/−173 was confirmed by DNA binding assays. Both YB-1 expression and CC chemokine ligand-5 (CCL5) mRNA expression were increased in neointimal versus medial smooth muscle cells, as analyzed by real-time polymerase chain reaction. Overexpression of YB-1 in smooth muscle cells (but not macrophages) enhanced CCL5 transcriptional activity in reporter assays, mRNA and protein expression, and CCL5-mediated monocyte arrest. Carotid arteries of hyperlipidemic apolipoprotein E-deficient mice were subjected to intraluminal transfection with a lentivirus encoding YB-1 short hairpin RNA or empty vector directly after wire injury. Double immunofluorescence revealed YB-1 expression in neointimal smooth muscle cells but not macrophages and colocalization with neointimal CCL5, which was downregulated by YB-1 short hairpin RNA. Neointima formation was decreased significantly after YB-1 knockdown compared with controls and was associated with a diminished content of lesional macrophages. A reduction of lesion formation by YB-1 knockdown was not observed in apolipoprotein E-deficient mice deficient in the CCL5 receptor CCR5 or after treatment with the CCL5 antagonist Met-RANTES, which indicates that YB-1 effects were dependent on CCL5. Conclusions— The transcriptional regulator YB-1 mediates CCL5 expression in smooth muscle cells and thereby contributes to neointimal hyperplasia, thus representing a novel target with which to limit vascular remodeling.

Y‐box protein‐1 is actively secreted through a non‐classical pathway and acts as an extracellular mitogen

Y‐box protein (YB)‐1 of the cold‐shock protein family functions in gene transcription and RNA processing. Extracellular functions have not been reported, but the YB‐1 staining pattern in inflammatory glomerular diseases, without adherence to cell boundaries, suggests an extracellular occurrence. Here, we show the secretion of YB‐1 by mesangial and monocytic cells after inflammatory challenges. It should be noted that YB‐1 was secreted through a non‐classical mode resembling that of the macrophage migration inhibitory factor. YB‐1 release requires ATP‐binding cassette transporters, and microvesicles protect YB‐1 from protease degradation. Two lysine residues in the YB‐1 carboxy‐terminal domain are crucial for its release, probably because of post‐translational modifications. The addition of purified recombinant YB‐1 protein to different cell types results in increased DNA synthesis, cell proliferation and migration. Thus, the non‐classically secreted YB‐1 has extracellular functions and exerts mitogenic as well as promigratory effects in inflammation.

Notch-3 receptor activation drives inflammation and fibrosis following tubulointerstitial kidney injury.

Kidney diseases impart a vast burden on affected individuals and the overall health care system. Progressive loss of renal parenchymal cells and functional decline following injury are often observed. Notch‐1 and ‐2 receptors are crucially involved in nephron development and contribute to inflammatory kidney diseases. We specifically determined the participation of receptor Notch‐3 following tubulointerstitial injury and in inflammatory responses. Here we show by heat map analyses that Notch‐3 transcripts are up‐regulated in human kidney diseases. A similar response was corroborated with kidney cells following TGF‐β exposure in vitro. The murine unilateral ureteral obstruction (UUO) model mirrors hallmarks of tubulointerstitial injury and damage. A subset of tubular and interstitial cells demonstrated up‐regulated Notch‐3 receptor expression in diseased animals. We hypothesized a relevance of Notch‐3 receptors for the chemotactic response. To address this question, animals with genetic ablation of receptor Notch‐3 were analysed following UUO. As a result, we found that Notch‐3‐deficient animals are protected from tubular injury and cell loss with significantly reduced interstitial collagen deposition. Monocytic cell infiltration was significantly reduced and retarded, likely due to abrogated chemokine synthesis. A cell model was set up that mimics enhanced receptor Notch‐3 expression and activation. Here a pro‐mitogenic response was seen with activated signalling in tubular cells and fibroblasts. In conclusion, Notch‐3 receptor fulfils non‐redundant roles in the inflamed kidney that may not be replaced by other Notch receptor family members. Thus, specific blockade of this receptor may be suitable as therapeutic option to delay progression of kidney disease. Copyright

Induction of progressive glomerulonephritis by podocyte-specific overexpression of platelet-derived growth factor-D

Platelet-derived growth factor-D (PDGF-D), normally expressed in podocytes, mediates mesangial cell proliferation in vivo. To study this further, we created transgenic mice with podocyte-specific overexpression of PDGF-D. Hemizygous mice were grossly indistinguishable from wild-type littermates through 11 months of age; however, hemizygous mice older than 4 weeks commonly exhibited increased cell proliferation within the glomerular tuft. Many hemizygous mice also developed widespread segmental glomerulosclerosis and focal extracapillary proliferation with fibrin/fibrinogen deposition, extensive tubulointerstitial damage, proteinuria, and renal insufficiency. Electron microscopy found focal foot process effacement. Renal mRNA expression of podocin and nephrin, as well as the number of glomerular WT-1-positive cells, were significantly reduced in hemizygous compared to wild-type mice, indicating loss and/or dedifferentation of podocytes. PDGF-A, -B, and both PDGF receptor chain mRNAs, fibronectin, type IV collagen, RANTES, MCP-1, and CCR-2 mRNAs were all increased in the renal cortex of PDGF-D transgenic mice. Only 8.5% of newborn mice were homozygous overexpressors exhibiting a mortality rate of 37% at 4 weeks. Thus, podocyte-specific overexpression of PDGF-D caused mesangioproliferative disease, glomerulosclerosis, and crescentic glomerulonephritis. Hence, podocyte-specific growth factor overexpression can induce paracrine mesangial cell proliferation upstream of the filtration flow.

Differential regulation of chemokine CCL5 expression in monocytes/macrophages and renal cells by Y-box protein-1

The Y-box protein-1 (YB-1) belongs to the family of cold shock proteins that have pleiotropic functions such as gene transcription, RNA splicing, and mRNA translation. YB-1 has a critical role in atherogenesis due to its regulatory effects on chemokine CCL5 (RANTES) gene transcription in vascular smooth muscle cells. Since CCL5 is a key mediator of kidney transplant rejection, we determined whether YB-1 is involved in allograft rejection by manipulating its expression. In human kidney biopsies, YB-1 transcripts were amplified 17-fold in acute and 21-fold in chronic allograft rejection with a close correlation between CCL5 and YB-1 mRNA expression in both conditions. Among three possible YB-1 binding sites in the CCL5 promoter, a critical element was mapped at -28/-10 bps. This site allowed up-regulation of CCL5 transcription in monocytic THP-1 and HUT78 T-cells and in human primary monocytes; however, it repressed transcription in differentiated macrophages. Conversely, YB-1 knockdown led to decreased CCL5 transcription and secretion in monocytic cells. We show that YB-1 is a cell-type specific regulator of CCL5 expression in infiltrating T-cells and monocytes/macrophages and acts as an adaptive controller of inflammation during kidney allograft rejection.

Extracellular YB-1 Blockade in Experimental Nephritis Upregulates Notch-3 Receptor Expression and Signaling

Background: Notch receptors are involved in kidney development and pathogenesis of inflammatory glomerular diseases. Given the secretion of Y-box (YB) protein-1 following cytokine stimulation and subsequent extracellular association with membrane receptor Notch-3 in vitro, we elucidated functional effects of YB-1 targeting on the Notch-3 signaling pathway. Methods: Rat mesangial cells were challenged with a monoclonal anti-YB-1 antibody (YB-1-mAb) and analyzed for YB-1 and Notch-3 expression. Notch-3 expression in mice with a targeted disruption of one YB-1 allele (YB-1+/d) was compared with their wild-type littermates. Furthermore, YB-1-mAb was applied during mesangioproliferative anti-Thy1.1 nephritis, and glomerular Notch-3, Notch target genes and YB-1 expression were analyzed by immunohistochemistry, quantitative real-time PCR and immunoblotting. Results: Upon challenge with YB-1-mAb, rat mesangial cells showed an increased expression of YB-1 and Notch-3 protein. Concordantly, we found a significant upregulation of Notch-3 expression in renal cells of YB-1+/d mice. YB-1-mAb treatment in anti-Thy1.1 nephritis resulted in enhanced mesangial Notch-3 expression and differential Notch target gene activation (HES2/Hey-2). Notably, YB-1 mRNA content did not differ between groups; however, glomerular YB-1 protein was significantly increased, suggesting a posttranslational mechanism. Conclusion: Extracellular targeting of YB-1 potently induces glomerular Notch-3 receptor expression, Notch signaling and YB-1 stabilization, most likely via an autoregulatory feedback mechanism.

Y-Box Binding Protein-1 Mediates Profibrotic Effects of Calcineurin Inhibitors in the Kidney

The immunosuppressive calcineurin inhibitors (CNIs) cyclosporine A (CsA) and tacrolimus are widely used in transplant organ recipients, but in the kidney allograft, they may cause tubulointerstitial as well as mesangial fibrosis, with TGF-β believed to be a central inductor. In this study, we report that the cold-shock protein Y-box binding protein-1 (YB-1) is a TGF-β independent downstream effector in CsA- as well as in tacrolimus- but not in rapamycin-mediated activation of rat mesangial cells (rMCs). Intracellular content of YB-1 is several-fold increased in MCs following CNI treatment in vitro and in vivo in mice. This effect ensues in a time-dependent manner, and the operative concentration range encompasses therapeutically relevant doses for CNIs. The effect of CNI on cellular YB-1 content is abrogated by specific blockade of translation, whereas retarding the transcription remains ineffective. The activation of rMCs by CNIs is accomplished by generation of reactive oxygen species. In contrast to TGF-β–triggered reactive oxygen species generation, hydrogen peroxide especially could be identified as a potent inductor of YB-1 accumulation. In line with this, hindering TGF-β did not influence CNI-induced YB-1 upregulation, whereas ERK/Akt pathways are involved in CNI-mediated YB-1 expression. CsA-induced YB-1 accumulation results in mRNA stabilization and subsequent generation of collagen. Our results provide strong evidence for a CNI-dependent induction of YB-1 in MCs that contributes to renal fibrosis via regulation of its own and collagen translation.

YB-1 Is an Early and Central Mediator of Bacterial and Sterile Inflammation In Vivo

In vitro studies identified Y-box–binding protein (YB)-1 as a key regulator of inflammatory mediators. In this study, we observed increased levels of secreted YB-1 in sera from sepsis patients. This led us to investigate the in vivo role of YB-1 in murine models of acute peritonitis following LPS injection, in sterile renal inflammation following unilateral ureteral obstruction, and in experimental pyelonephritis. LPS injection enhanced de novo secretion of YB-1 into the urine and the peritoneal fluid of LPS-treated mice. Furthermore, we could demonstrate a significant, transient upregulation and posttranslational modification (phosphorylation at serine 102) of YB-1 in renal and inflammatory cells. Increased renal cytoplasmic YB-1 amounts conferred enhanced expression of proinflammatory chemokines CCL2 and CCL5. Along these lines, heterozygous YB-1 knockout mice (YB-1+/d) that display 50% reduced YB-1 levels developed significantly lower responses to both LPS and sterile inflammation induced by unilateral ureteral obstruction. This included diminished immune cell numbers due to impaired migration propensities and reduced chemokine expression. YB-1+/d mice were protected from LPS-associated mortality (20% mortality on day 3 versus 80% in wild-type controls); however, immunosuppression in YB-1+/d animals resulted in 50% mortality. In conclusion, our findings identify YB-1 as a major, nonredundant mediator in both systemic and local inflammatory responses.

Cold shock Y-box protein-1 proteolysis autoregulates its transcriptional activities

BackgroundThe Y-box protein-1 (YB-1) fulfills pleiotropic functions relating to gene transcription, mRNA processing, and translation. It remains elusive how YB-1 shuttling into the nuclear and cytoplasmic compartments is regulated and whether limited proteolysis by the 20S proteasome releases fragments with distinct function(s) and subcellular distribution(s).ResultsTo address these questions, mapping of domains responsible for subcellular targeting was performed. Three nuclear localization signals (NLS) were identified. NLS-1 (aa 149-156) and NLS-2 (aa 185-194) correspond to residues with unknown function(s), whereas NLS-3 (aa 276-292) matches with a designated multimerization domain. Nuclear export signal(s) were not identified. Endoproteolytic processing by the 20S proteasome before glycine 220 releases a carboxy-terminal fragment (CTF), which localized to the nucleus, indicating that NLS-3 is operative. Genotoxic stress induced proteolytic cleavage and nuclear translocation of the CTF. Co-expression of the CTF and full-length YB-1 resulted in an abrogated transcriptional activation of the MMP-2 promoter, indicating an autoregulatory inhibitory loop, whereas it fulfilled similar trans-repressive effects on the collagen type I promoter.ConclusionCompartmentalization of YB-1 protein derivatives is controlled by distinct NLS, one of which targets a proteolytic cleavage product to the nucleus. We propose a model for an autoregulatory negative feedback loop that halts unlimited transcriptional activation.

Role of cold shock Y-box protein-1 in inflammation, atherosclerosis and organ transplant rejection.

Chemokines (chemoattractant cytokines) are crucial regulators of immune cell extravasation from the bloodstream into inflamed tissue. Dysfunctional regulation and perpetuated chemokine gene expression are linked to progressive chronic inflammatory diseases and, in respect to transplanted organs, may trigger graft rejection. RANTES (regulated upon activation, normal T cell expressed and secreted (also known as CCL5)) is a model chemokine with relevance in numerous inflammatory diseases where the innate immune response predominates. Transcription factor Y-box binding protein-1 (YB-1) serves as a trans-regulator of CCL5 gene transcription in vascular smooth muscle cells and leucocytes. This review provides an update on YB-1 as a mediator of inflammatory processes and focuses on the role of YB-1 in CCL5 expression in diseases with monocytic cell infiltrates, albeit acute or chronic. Paradigms of such diseases encompass atherosclerosis and transplant rejection where cold shock protein YB-1 takes a dominant role in transcriptional regulation.