Eva Dizin

NADPH-Oxidase 4 Protects against Kidney Fibrosis during Chronic Renal Injury

NADPH oxidases synthesize reactive oxygen species that may participate in fibrosis progression. NOX4 and NOX2 are NADPH oxidases expressed in the kidneys, with the former being the major renal isoform, but their contribution to renal disease is not well understood. Here, we used the unilateral urinary obstruction model of chronic renal injury to decipher the role of these enzymes using wild-type, NOX4-, NOX2-, and NOX4/NOX2-deficient mice. Compared with wild-type mice, NOX4-deficient mice exhibited more interstitial fibrosis and tubular apoptosis after obstruction, with lower interstitial capillary density and reduced expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in obstructed kidneys. Furthermore, NOX4-deficient kidneys exhibited increased oxidative stress. With NOX4 deficiency, renal expression of other NOX isoforms was not altered but NRF2 protein expression was reduced under both basal and obstructed conditions. Concomitant deficiency of NOX2 did not modify the phenotype exhibited by NOX4-deficient mice after obstruction. NOX4 silencing in a mouse collecting duct (mCCD(cl1)) cell line increased TGF-β1-induced apoptosis and decreased NRF2 protein along with expression of its target genes. In addition, NOX4 silencing decreased hypoxia-inducible factor-1α and expression of its target genes in response to hypoxia. In summary, these results demonstrate that the absence of NOX4 promotes kidney fibrosis, independent of NOX2, through enhanced tubular cell apoptosis, decreased microvascularization, and enhanced oxidative stress. Thus, NOX4 is crucial for the survival of kidney tubular cells under injurious conditions.

Common Variation at BARD1 Results in the Expression of an Oncogenic Isoform That Influences Neuroblastoma Susceptibility and Oncogenicity

The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study, we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1β. In neuroblastoma cells, silencing of BARD1β showed genotype-specific cytotoxic effects, including decreased substrate-adherence, anchorage-independence, and foci growth. In established murine fibroblasts, overexpression of BARD1β was sufficient for neoplastic transformation. BARD1β stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1β as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1β with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma.

Distinct Roles of BARD1 Isoforms in Mitosis: Full-Length BARD1 Mediates Aurora B Degradation, Cancer-Associated BARD1β Scaffolds Aurora B and BRCA2

The BRCA1-associated ring domain protein 1 (BARD1) interacts with BRCA1 via its RING finger domain. The BARD1-BRCA1 complex participates in DNA repair, cell cycle control, genomic stability, and mitotic spindle formation through its E3 ubiquitin ligase activity. Cancer cells express several BARD1 protein isoforms, including the RING finger-deficient variant BARD1beta. Here, we show that BARD1 has BRCA1-dependent and BRCA1-independent functions in mitosis. BARD1, but not BRCA1, localizes to the midbody at telophase and cytokinesis, where it colocalizes with Aurora B. The 97-kDa full-length (FL) BARD1 coimmunoprecipates with BRCA1, but the 82-kDa BARD1beta coimmunoprecipitates with Aurora B and BRCA2. We used selective small interfering RNAs to distinguish the functions of FL BARD1 and BARD1beta. Depletion of FL BARD1 had only minor effects on cell growth and did not abolish midbody localization of BARD1 staining, but resulted in massive up-regulation of Aurora B. In contrast, suppression of FL BARD1 and BARD1beta led to growth arrest and correlated with various mitotic defects and disappearance of midbody localization of BARD1 staining. Our data suggest a novel function of FL BARD1 in Aurora B ubiquitination and degradation, opposing a proproliferative function of BARD1beta in scaffolding Aurora B and BRCA2. Thus, loss of FL BARD1 and up-regulation of Aurora B, as observed in cancer cells, can be explained by an imbalance of FL BARD1 and BARD1beta.

Oncogenic BARD1 Isoforms Expressed in Gynecological Cancers

BARD1 is required for protein stability and tumor suppressor functions of BRCA1, which depend on the ubiquitin ligase activity of the BRCA1-BARD1 heterodimer. The NH(2)-terminal RING domains of both proteins act as interaction modules and form a ubiquitin ligase, which has functions in DNA repair, cell cycle checkpoint regulation, and mitosis. Interestingly, up-regulated expression of truncated BARD1 isoforms was found to be associated with poor prognosis in breast and ovarian cancers and, in a hormonally regulated fashion, in the human cytotrophoblast, a cell type with properties reminiscent of cancer cells. We therefore performed reverse transcription-PCR to determine the structure of BARD1 isoforms in cell lines derived from hormone-dependent and hormone-independent cancers. We found a specific combination of isoforms, generated by differential splicing and alternative transcription initiation, mostly lacking the BRCA1 interaction domain, in gynecologic but not hematologic cancer cell lines. To investigate the prevalence of BARD1 isoforms in tumors, we applied immunohistochemistry to ovarian cancers, using antibodies distinguishing full-length BARD1 and isoforms. Expression of NH(2) terminally truncated BARD1 was correlated with advanced stage of cancer, and expression of spliced isoforms was typical for clear cell carcinoma, the ovarian cancer with worst prognosis, suggesting a role of BARD1 isoforms in cancer progression. To challenge this hypothesis, we silenced BARD1 isoforms in ovarian cancer cells that lacked wild-type BARD1 by siRNA interference, which led to a complete proliferation arrest. Thus, BARD1 isoform expression is required for cancer cell proliferation, which is compatible with the notion that BARD1 isoforms act as cancer maintenance genes.

Negative feedback loop of BRCA1–BARD1 ubiquitin ligase on estrogen receptor alpha stability and activity antagonized by cancer-associated isoform of BARD1

Estrogen is involved in breast cancer risk, which is increased for BRCA1 mutation carriers, suggesting a role for BRCA1 in estrogen signaling. BRCA1 exerts its function through forming an E3 ubiquitin ligase with BARD1. We report that the estrogen receptor alpha is a target of the BRCA1-BARD1 ubiquitin ligase in vivo. BRCA1 and BARD1 are required for estrogen receptor alpha ubiquitination and degradation, and repression of either one leads to ERalpha accumulation, suggesting a feedback loop between BRCA1-BARD1 and estrogen receptor alpha, since BRCA1 and BARD1 are induced by estrogen receptor alpha. While the ubiquitin ligase activity maps to the N-terminal RING finger domains of BRCA1 and BARD1, we demonstrate that the BARD1 C-terminus is important for target recognition. Furthermore, a BARD1 isoform lacking the RING domain binds and stabilizes estrogen receptor alpha. Thus deficiencies of BRCA1 or BARD1 and/or upregulation of BARD1 isoforms lead to estrogen receptor alpha upregulation, providing a functional link between BRCA1 deficiency, estrogen signaling, and tumorigenesis.

Proteinuria Increases Plasma Phosphate by Altering Its Tubular Handling

Proteinuria and hyperphosphatemia are cardiovascular risk factors independent of GFR. We hypothesized that proteinuria induces relative phosphate retention via increased proximal tubule phosphate reabsorption. To test the clinical relevance of this hypothesis, we studied phosphate handling in nephrotic children and patients with CKD. Plasma fibroblast growth factor 23 (FGF-23) concentration, plasma phosphate concentration, and tubular reabsorption of phosphate increased during the proteinuric phase compared with the remission phase in nephrotic children. Cross-sectional analysis of a cohort of 1738 patients with CKD showed that albuminuria≥300 mg/24 hours is predictive of higher phosphate levels, independent of GFR and other confounding factors. Albuminuric patients also displayed higher plasma FGF-23 and parathyroid hormone levels. To understand the molecular mechanisms underlying these observations, we induced glomerular proteinuria in two animal models. Rats with puromycin-aminonucleoside-induced nephrotic proteinuria displayed higher renal protein expression of the sodium-phosphate co-transporter NaPi-IIa, lower renal Klotho protein expression, and decreased phosphorylation of FGF receptor substrate 2α, a major FGF-23 receptor substrate. These findings were confirmed in transgenic mice that develop nephrotic-range proteinuria resulting from podocyte depletion. In vitro, albumin did not directly alter phosphate uptake in cultured proximal tubule OK cells. In conclusion, we show that proteinuria increases plasma phosphate concentration independent of GFR. This effect relies on increased proximal tubule NaPi-IIa expression secondary to decreased FGF-23 biologic activity. Proteinuria induces elevation of both plasma phosphate and FGF-23 concentrations, potentially contributing to cardiovascular disease.

Sodium Transport Is Modulated by p38 Kinase–Dependent Cross-Talk between ENaC and Na,K-ATPase in Collecting Duct Principal Cells

In relation to dietary Na(+) intake and aldosterone levels, collecting duct principal cells are exposed to large variations in Na(+) transport. In these cells, Na(+) crosses the apical membrane via epithelial Na(+) channels (ENaC) and is extruded into the interstitium by Na,K-ATPase. The activity of ENaC and Na,K-ATPase must be highly coordinated to accommodate variations in Na(+) transport and minimize fluctuations in intracellular Na(+) concentration. We hypothesized that, independent of hormonal stimulus, cross-talk between ENaC and Na,K-ATPase coordinates Na(+) transport across apical and basolateral membranes. By varying Na(+) intake in aldosterone-clamped rats and overexpressing γ-ENaC or modulating apical Na(+) availability in cultured mouse collecting duct cells, enhanced apical Na(+) entry invariably led to increased basolateral Na,K-ATPase expression and activity. In cultured collecting duct cells, enhanced apical Na(+) entry increased the basolateral cell surface expression of Na,K-ATPase by inhibiting p38 kinase-mediated endocytosis of Na,K-ATPase. Our results reveal a new role for p38 kinase in mediating cross-talk between apical Na(+) entry via ENaC and its basolateral exit via Na,K-ATPase, which may allow principal cells to maintain intracellular Na(+) concentrations within narrow limits.

NADPH Oxidase 4 Deficiency Reduces Aquaporin-2 mRNA Expression in Cultured Renal Collecting Duct Principal Cells via Increased PDE3 and PDE4 Activity

The final control of renal water reabsorption occurs in the collecting duct (CD) and relies on regulated expression of aquaporin-2 (AQP2) in principal CD cells. AQP2 transcription is primarily induced by type 2 vasopressin receptor (V2R)-cAMP-protein kinase A (PKA) signaling but also by other factors, including TonEBP and NF-κB. NAPDH oxidase 4 (NOX4) represents a major source of reactive oxygen species (ROS) in the kidney. Because NOX-derived ROS may alter PKA, TonEBP and NF-κB activity, we examined the effects of NOX4 depletion on AQP2 expression. Depleted NOX4 expression by siRNA (siNOX4) in mpkCCDcl4 cells attenuated increased AQP2 mRNA expression by arginine vasopressin (AVP) but not by hypertonicity, which induces both TonEBP and NF-κB activity. AVP-induced AQP2 expression was similarly decreased by the flavoprotein inhibitor diphenyleneiodonium. siNOX4 altered neither TonEBP nor NF-κB activity but attenuated AVP-inducible cellular cAMP concentration, PKA activity and CREB phosphorylation as well as AQP2 mRNA expression induced by forskolin, a potent activator of adenylate cyclase. The repressive effect of siNOX4 on AVP-induced AQP2 mRNA expression was abolished by the non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and was significantly decreased by selective PDE antagonists cilostamide and rolipram, but not vinpocetine, which respectively target PDE3, PDE4 and PDE1. Thus, by inhibiting PDE3 and PDE4 activity NOX4-derived ROS may contribute to V2R-cAMP-PKA signaling and enhance AQP2 transcription.

Albuminuria induces a proinflammatory and profibrotic response in cortical collecting ducts via the 24p3 receptor

Albuminuria is strongly associated with progressive kidney tubulo-interstitial damage and chronic kidney disease (CKD) progression. In proteinuric nephropathies, albumin reabsorption by the proximal tubule is saturated and the distal nephron is exposed to high concentrations of luminal albumin that may produce adverse effects. Since proximal tubular cells exposed to albuminuria exhibit a proinflammatory and profibrotic response, we assessed the effect of albuminuria in the collecting duct (CD). With the use of kidney sections and isolated cortical CDs (CCDs) from puromycin-aminonucleoside-induced nephrotic rats (PAN rats) exhibiting proteinuria, immunofluorescence microscopy revealed internalized albumin in CD cells. In these proteinuric rats, increased expression levels of cytokines and profibrotic signaling markers were detected in isolated CCDs and bands of inflammatory fibrosis could be observed around CDs. Albumin endocytosis was confirmed by FITC-albumin uptake in cultured murine CCD (mCCDcl1) cells. Exposure of mCCDcl1 cells to albumin induced NF-κB activation as assessed by luciferase reporter gene assay, nuclear translocation of NF-κB p65 subunit, and increased NF-κB target gene expression. Moreover, albuminuria-like condition results in transforming growth factor-β1 (TGF-β1) overexpression and the upregulation of profibrotic signaling markers such as Snail or vimentin via an autocrine mechanism. In mCCDcl1 cells, neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin-2/24p3 receptor (24p3R) mediates albumin endocytosis as well as activation of NF-κB and TGF-β1 signaling pathways. Therefore, CD may play a key role in initiation and/or progression of inflammation and fibrosis in response to proteinuria.

Caspase-dependent BRCA1 cleavage facilitates chemotherapy-induced apoptosis

BRCA1 acts as a tumor suppressor gene, and germ-line mutations in this gene are found in a large proportion of families with breast and ovarian cancers. The BRCA1 protein has been implicated in several cellular processes, such as transcription regulation, DNA responses to DNA damage signals, cell cycle control, and apoptosis. Apoptosis plays a critical role in radiation- and chemotherapy-induced cytotoxicity, and its impairment contributes to resistance to tumor treatments. In an attempt to elucidate the role of BRCA1 in apoptosis, we examined the response to chemotherapeutic drugs of cells expressing physiological levels of BRCA1 protein. We showed that chemotherapy-induced apoptosis leads to a caspase-mediated cleavage of BRCA1. We then showed that the BRCA1-p90 cleavage product is mainly localized in the cytoplasm. Finally, we demonstrated that cancer-associated mutations affecting the BRCT tandem repeat abolish its pro-apoptotic function. The data presented here provide new insight into the role of endogenous BRCA1 as a mediator of apoptosis and show that BRCA1 functions as a molecular determinant of response to a range of cytotoxic chemotherapeutic agents.