Activation of the Hypoxia-Inducible Factor Pathway Inhibits Epithelial Sodium Channel-Mediated Sodium Transport in Collecting Duct Principal Cells.

BACKGROUND: Active sodium reabsorption is the major factor influencing renal oxygen consumption and production of reactive oxygen species (ROS). Increased sodium reabsorption uses more oxygen, which may worsen medullary hypoxia and produce more ROS via enhanced mitochondrial ATP synthesis. Both mechanisms may activate the hypoxia-inducible factor (HIF) pathway. Because the collecting duct is exposed to low oxygen pressure and variations of active sodium transport, we assessed whether the HIF pathway controls epithelial sodium channel (ENaC)-dependent sodium transport. METHODS: We investigated HIF's effect on ENaC expression in mpkCCD cl4 cells (a model of collecting duct principal cells) using real-time PCR and western blot and ENaC activity by measuring amiloride-sensitive current. We also assessed the effect of hypoxia and sodium intake on abundance of kidney sodium transporters in wild-type and inducible kidney tubule-specific Hif1α knockout mice. RESULTS: In cultured cells, activation of the HIF pathway by dimethyloxalylglycine or hypoxia inhibited sodium transport and decreased expression of ß ENaC and γ ENaC, as well as of Na,K-ATPase. HIF1 α silencing increased ß ENaC and γ ENaC expression and stimulated sodium transport. A constitutively active mutant of HIF1 α produced the opposite effect. Aldosterone and inhibition of the mitochondrial respiratory chain slowly activated the HIF pathway, suggesting that ROS may also activate HIF. Decreased γ ENaC abundance induced by hypoxia in normal mice was abolished in Hif1α knockout mice. Similarly, Hif1α knockout led to increased γ ENaC abundance under high sodium intake. CONCLUSIONS: This study reveals that γ ENaC expression and activity are physiologically controlled by the HIF pathway, which may represent a negative feedback mechanism to preserve oxygenation and/or prevent excessive ROS generation under increased sodium transport.

Interaction between Epithelial Sodium Channel γ-Subunit and Claudin-8 Modulates Paracellular Sodium Permeability in Renal Collecting Duct.

BACKGROUND: Water and solute transport across epithelia can occur via the transcellular or paracellular pathways. Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. In the renal collecting duct, which is a typical absorptive tight epithelium, coordination between transcellular sodium reabsorption and paracellular permeability may prevent the backflow of reabsorbed sodium to the tubular lumen along a steep electrochemical gradient. METHODS: To investigate whether transcellular sodium transport controls tight-junction composition and paracellular permeability via modulating expression of the transmembrane protein claudin-8, we used cultured mouse cortical collecting duct cells to see how overexpression or silencing of epithelial sodium channel (ENaC) subunits and claudin-8 affect paracellular permeability. We also used conditional kidney tubule-specific knockout mice lacking ENaC subunits to assess the ENaC's effect on claudin-8 expression. RESULTS: Overexpression or silencing of the ENaC γ-subunit was associated with parallel and specific changes in claudin-8 abundance. Increased claudin-8 abundance was associated with a reduction in paracellular permeability to sodium, whereas decreased claudin-8 abundance was associated with the opposite effect. Claudin-8 overexpression and silencing reproduced these functional effects on paracellular ion permeability. Conditional kidney tubule-specific ENaC γ-subunit knockout mice displayed decreased claudin-8 expression, confirming the cell culture experiments' findings. Importantly, ENaC ß-subunit or α-subunit silencing or kidney tubule-specific ß-ENaC or α-ENaC knockout mice did not alter claudin-8 abundance. CONCLUSIONS: Our data reveal the specific coupling between ENaC γ-subunit and claudin-8 expression. This coupling may play an important role in preventing the backflow of reabsorbed solutes and water to the tubular lumen, as well as in coupling paracellular and transcellular sodium permeability.

Time-course of sodium transport along the nephron in nephrotic syndrome: The role of potassium.

The mechanism of sodium retention and its location in kidney tubules may vary with time in nephrotic syndrome (NS). We studied the mechanisms of sodium retention in transgenic POD-ATTAC mice, which display an inducible podocyte-specific apoptosis. At day 2 after the induction of NS, the increased abundance of NHE3 and phosphorylated NCC in nephrotic mice compared with controls suggest that early sodium retention occurs mainly in the proximal and distal tubules. At day 3, the abundance of NHE3 normalized, phosphorylated NCC levels decreased, and cleavage and apical localization of γ-ENaC increased in nephrotic mice. These findings indicate that sodium retention shifted from the proximal and distal tubules to the collecting system. Increased cleavage and apical localization of γ-ENaC persisted at day 5 in nephrotic mice when hypovolemia resolved and steady-state was reached. Sodium retention and γ-ENaC cleavage were independent of the increased plasma levels of aldosterone. Nephrotic mice displayed decreased glomerular filtration rate and urinary potassium excretion associated with hyperkaliemia at day 3. Feeding nephrotic mice with a low potassium diet prevented hyperkaliemia, γ-ENaC cleavage, and led to persistent increased phosphorylation of NCC. These results suggest that potassium homeostasis is a major determinant of the tubular site of sodium retention in nephrotic mice.

Liver Glutamate Dehydrogenase Controls Whole-Body Energy Partitioning Through Amino Acid-Derived Gluconeogenesis and Ammonia Homeostasis.

Ammonia detoxification and gluconeogenesis are major hepatic functions mutually connected through amino acid metabolism. The liver is rich in glutamate dehydrogenase (GDH) that catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate and ammonia, thus bridging amino acid-to-glucose pathways. Here we generated inducible liver-specific GDH-knockout mice (HepGlud1-/- ) to explore the role of hepatic GDH on metabolic homeostasis. Investigation of nitrogen metabolism revealed altered ammonia homeostasis in HepGlud1-/- mice characterized by increased circulating ammonia associated with reduced detoxification process into urea. The abrogation of hepatic GDH also modified energy homeostasis. In the fasting state, HepGlud1-/- mice could barely produce glucose in response to alanine due to impaired liver gluconeogenesis. Compared with control mice, lipid consumption in HepGlud1-/- mice was favored over carbohydrates as a compensatory energy fuel. The changes in energy partitioning induced by the lack of liver GDH modified the circadian rhythm of food intake. Overall, this study demonstrates the central role of hepatic GDH as a major regulator for the maintenance of ammonia and whole-body energy homeostasis.

Dietary sodium induces a redistribution of the tubular metabolic workload.

KEY POINTS: Body Na+ content is tightly controlled by regulated urinary Na+ excretion. The intrarenal mechanisms mediating adaptation to variations in dietary Na+ intake are incompletely characterized. We confirmed and expanded observations in mice that variations in dietary Na+ intake do not alter the glomerular filtration rate but alter the total and cell-surface expression of major Na+ transporters all along the kidney tubule. Low dietary Na+ intake increased Na+ reabsorption in the proximal tubule and decreased it in more distal kidney tubule segments. High dietary Na+ intake decreased Na+ reabsorption in the proximal tubule and increased it in distal segments with lower energetic efficiency. The abundance of apical transporters and Na+ delivery are the main determinants of Na+ reabsorption along the kidney tubule. Tubular O2 consumption and the efficiency of sodium reabsorption are dependent on sodium diet. ABSTRACT: Na+ excretion by the kidney varies according to dietary Na+ intake. We undertook a systematic study of the effects of dietary salt intake on glomerular filtration rate (GFR) and tubular Na+ reabsorption. We examined the renal adaptive response in mice subjected to 7 days of a low sodium diet (LSD) containing 0.01% Na+ , a normal sodium diet (NSD) containing 0.18% Na+ and a moderately high sodium diet (HSD) containing 1.25% Na+ . As expected, LSD did not alter measured GFR and increased the abundance of total and cell-surface NHE3, NKCC2, NCC, α-ENaC and cleaved γ-ENaC compared to NSD. Mathematical modelling predicted that tubular Na+ reabsorption increased in the proximal tubule but decreased in the distal nephron because of diminished Na+ delivery. This prediction was confirmed by the natriuretic response to diuretics targeting the thick ascending limb, the distal convoluted tubule or the collecting system. On the other hand, HSD did not alter measured GFR but decreased the abundance of the aforementioned transporters compared to NSD. Mathematical modelling predicted that tubular Na+ reabsorption decreased in the proximal tubule but increased in distal segments with lower transport efficiency with respect to O2 consumption. This prediction was confirmed by the natriuretic response to diuretics. The activity of the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) was related to the changes in tubular Na+ reabsorption. Our data show that fractional Na+ reabsorption is distributed differently according to dietary Na+ intake and induces changes in tubular O2 consumption and sodium transport efficiency.

Coordinated Control of ENaC and Na+,K+-ATPase in Renal Collecting Duct.

Tubular reabsorption of filtered sodium is tightly controlled to maintain body volume homeostasis. The rate of sodium transport by collecting duct (CD) cells varies widely in response to dietary sodium intake, GFR, circulating hormones, neural signals, and local regulatory factors. Reabsorption of filtered sodium by CD cells occurs via a two-step process. First, luminal sodium crosses the apical plasma membrane along its electrochemical gradient through epithelial sodium channels (ENaC). Intracellular sodium is then actively extruded into the interstitial space by the Na(+),K(+)-ATPase located along the basolateral membrane. Mismatch between sodium entry and exit induces variations in sodium intracellular concentration and cell volume that must be maintained within narrow ranges for control of vital cell functions. Therefore, renal epithelial cells display highly coordinated apical and basolateral sodium transport rates. We review evidence from experiments conducted in vivo and in cultured cells that indicates aldosterone and vasopressin, the two major hormones regulating sodium reabsorption by CD, generate a coordinated stimulation of apical ENaC and basolateral Na(+),K(+)-ATPase. Moreover, we discuss evidence suggesting that variations in sodium entry per se induce a coordinated change in Na(+),K(+)-ATPase activity through the signaling of protein kinases such as protein kinase A and p38 mitogen-activated protein kinase.

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.

Epithelial sodium channel abundance is decreased by an unfolded protein response induced by hyperosmolality.

Large shifts of osmolality occur in the kidney medulla as part of the urine concentrating mechanism. Hyperosmotic stress profoundly challenges cellular homeostasis and induces endoplasmic reticulum (ER) stress. Here, we examined the unfolded protein response (UPR) in hyperosmotically-challenged principal cells of the kidney collecting duct (CD) and show its relevance in controlling epithelial sodium channel (ENaC) abundance, responsible for the final adjustment of Na(+) excretion. Dehydration increases medullary but not cortical osmolality. Q-PCR analysis of microdissected CD of water-deprived mice revealed increased aquaporin-2 (AQP2) expression in outer medullary and cortical CD while ENaC abundance decreased in outer medullary but not cortical CD. Immunoblotting, Q-PCR and immunofluorescence revealed that hyperosmolality induced a transient ER stress-like response both ex vivo and in cultured CD principal cells and increased activity of the canonical UPR mediators PERK and ATF6. Both hyperosmolality and chemical induction of ER stress decreased ENaC expression in vitro. ENaC depletion by either stimulus was abolished by transcriptional inhibition and by the chemical chaperone 4-phenylbutyric acid and was partly abrogated by either PERK or ATF6 silencing. Our data suggest that induction of the UPR by hyperosmolality may help preserve body fluid homeostasis under conditions of dehydration by uncoupling AQP2 and ENaC abundance in outer medullary CD.

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.

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.

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-ß1 (TGF-ß1) 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-ß1 signaling pathways. Therefore, CD may play a key role in initiation and/or progression of inflammation and fibrosis in response to proteinuria.

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-ß1-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, BARD1ß. In neuroblastoma cells, silencing of BARD1ß showed genotype-specific cytotoxic effects, including decreased substrate-adherence, anchorage-independence, and foci growth. In established murine fibroblasts, overexpression of BARD1ß was sufficient for neoplastic transformation. BARD1ß 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 BARD1ß 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 BARD1ß with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma.

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.

Distinct roles of BARD1 isoforms in mitosis: full-length BARD1 mediates Aurora B degradation, cancer-associated BARD1beta 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.

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.

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.

BRCA1 interacts with poly(A)-binding protein: implication of BRCA1 in translation regulation.

BRCA1 has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair, cell cycle control, and apoptosis. We identified poly(A)-binding protein 1 (PABP) as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed the interaction by in vitro assays and coimmunoprecipitation in mammalian cells. Endogenous interaction between BRCA1 and PABP was also observed. This interaction was abolished by BRCA1 cancer-associated mutations, suggesting that it may be physiologically relevant. Deletion mapping demonstrated that the RNA recognition motifs 1-4 region of PABP is required to mediate the interaction with BRCA1. To understand the biological function of the BRCA1-PABP complex, we sought to determine whether BRCA1 is a modulator of translation. We showed here that inhibition of endogenous BRCA1 using a small interfering RNA-based approach decreased protein synthesis. Conversely, overexpression of BRCA1 activated translation. Using a RNA transfection approach, we clearly showed that BRCA1 modulates translation, independently of any transcriptional activity. The data presented here suggest that BRCA1 modulates protein synthesis via its interaction with PABP, providing a novel mechanism by which BRCA1 may exert its tumor suppressor function.

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1.

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.

BRCA1 affects lipid synthesis through its interaction with acetyl-CoA carboxylase.

Germ line alterations in BRCA1 (breast cancer susceptibility gene 1) are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 acts as a scaffold protein implicated in multiple cellular functions, such as transcription, DNA repair, and ubiquitination. However, the molecular mechanisms responsible for tumorigenesis are not yet fully understood. We have recently demonstrated that BRCA1 interacts in vivo with acetyl coenzyme A carboxylase alpha (ACCA) through its tandem of BRCA1 C terminus (BRCT) domains. To understand the biological function of the BRCA1.ACCA complex, we sought to determine whether BRCA1 is a regulator of lipogenesis through its interaction with ACCA. We showed here that RNA inhibition-mediated down-regulation of BRCA1 expression induced a marked increase in the fatty acid synthesis. We then delineated the biochemical characteristics of the complex and found that BRCA1 interacts solely with the phosphorylated and inactive form of ACCA (P-ACCA). Finally, we demonstrated that BRCA1 affects lipid synthesis by preventing P-ACCA dephosphorylation. These results suggest that BRCA1 affects lipogenesis through binding to P-ACCA, providing a new mechanism by which BRCA1 may exert a tumor suppressor function.