Calcineurin-mediated YB-1 dephosphorylation regulates CCL5 expression during monocyte differentiation.

Y-box (YB) protein-1 serves as a master regulator in gene transcription and mRNA translation. YB-1 itself is regulated at various levels, e.g. through post-translational modifications. In our previous work, we identified RANTES/CCL5 as a transcriptional target of YB-1. We previously demonstrated that YB-1 protein is transiently up-regulated during monocyte/macrophage differentiation evidenced in monocytic cells (THP-1 cells) that were differentiated using phorbol myristate acetate (PMA). Here we provide evidence that YB-1 phosphorylation, specifically at its serine residue 102 (Ser-102), increases early on in THP-1 cells following PMA treatment as well as in differentiated primary human monocytes. This process is mediated through the Akt signaling pathway. Ser-102-phosphorylated YB-1 displays stronger binding affinity and trans-activating capacity at the CCL5 gene promoter. Notably, Ser-102-phosphorylated YB-1 disappears at later stages of the monocyte/macrophage differentiation process. We demonstrate that serine-threonine phosphatase calcineurin (CN) dephosphorylates YB-1 preventing it from binding to and trans-activating the CCL5 promoter. Co-immunoprecipitation assays prove a direct YB-1/CN interaction. Furthermore, analyses in kidney tissues from mice that were treated with the CN inhibitor cyclosporine A revealed an in vivo effect of CN on the YB-1 phosphorylation status. We conclude that YB-1 phosphorylation at Ser-102 is an important prerequisite for CCL5 promoter activation during macrophage differentiation. Our findings point to a critical role of YB-1 in the resolution of inflammatory processes which may largely be due to CN-mediated dephosphorylation.

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.

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-ß 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.

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-ß 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-ß 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-ß-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-ß 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.

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.

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.