Deletion of KS-WNK1 promotes NCC activation by increasing WNK1/4 abundance.

Dietary potassium deficiency causes stimulation of sodium reabsorption leading to increased risk in blood pressure elevation. The distal convoluted tubule is the main rheostat linking plasma K+ levels to the activity of the Na-Cl cotransporter (NCC). This occurs through basolateral membrane potential sensing by Kir4.1/5.1; decrease in intracellular Cl-; activation of WNK4, interaction and phosphorylation of Ste20/SPS1-related Proline/Alanine-rich Kinase (SPAK); binding of the calcium-binding protein 39 (cab39) adaptor protein to SPAK leading to its trafficking to the apical membrane; and SPAK binding, phosphorylating, and activating NCC. As Kidney-Specific With-No-Lysine (K) Kinase 1 (WNK1) isoform (KS-WNK1) is another participant in this pathway, we examined its function in NCC regulation. We eliminated KS-WNK1 specifically in the DCT and demonstrated increased expression of WNK4 and L-WNK1 and increased phosphorylation of NCC. As in other KS-WNK1 models, the mice are not hyperkalemic. While wild-type mice under low dietary K+ conditions demonstrated increased NCC phosphorylation, the phosphorylation levels of the transporter, already high in the KS-WNK1, did not change under the low K+ diet. Thus, in the absence of KS-WNK1 the transporter has lost its sensitivity to low plasma K+. We also show that under low K+ conditions, in the absence of KS-WNK1, there is no formation of WNK bodies. These bodies are observed in adjacent segments, not affected by the targeting of KS-WNK1. As our data are overall consistent with those of the global KS-WNK1 knockout, they indicate that the DCT is the predominant segment affecting the salt transport regulated by KS-WNK1.

NKCC1 in human diseases: is the SLC12A2 gene haploinsufficient?

Mutations in the SLC12A2 gene, which encodes the Na-K-2Cl cotransporter-1 (NKCC1), are linked to various conditions such as neurodevelopmental deficits, deafness, and fluid secretion in different epithelia. Cases of complete NKCC1 deficiency in young patients are straightforward, leading to clinical presentations that overlap with the phenotypes observed in NKCC1 knockout mouse models. However, cases involving deleterious variants in one allele are more difficult, as the clinical presentation is variable, and the cause-effect relationship is not always clear. For instance, we worked on a single patient's case from multiple angles and published six related papers to convince ourselves of the cause-and-effect relationship between her NKCC1 mutation and her clinical presentations. The cluster of mutations in a small portion of the carboxyl terminus and its association with deafness point to a cause-and-effect relationship, even if the molecular mechanism is unknown. Overall, the preponderance of evidence suggests that the SLC12A2 gene is a human disease-causing and likely haploinsufficient gene that requires further investigation.

Bicarbonate is the primary inducer of KCC3a expression in renal cortical B-type intercalated cells.

A primary function of intercalated cells in the distal tubule of the kidney is to maintain pH homeostasis. For example, type B intercalated cells secrete bicarbonate largely through the action of the apical Cl-/HCO3- exchanger, pendrin, which helps correct metabolic alkalosis. Since both the K-Cl cotransporter, KCC3a and pendrin colocalize to the apical region of type B and non-A, non-B intercalated cells and since both are upregulated in models of metabolic alkalosis, such as with dietary NaHCO3 loading, we raised the possibility that apical KCC3a facilitates pendrin-mediated bicarbonate secretion, such as through apical Cl- recycling. The purpose of this study was to determine if KCC3a abundance changes through intake of bicarbonate alone or through bicarbonate plus its accompanying cation, and if it requires a direct interaction with pendrin or the renin-angiotensin-aldosterone system. We observed that KCC3a protein abundance, but not mRNA, increases in a mouse model of metabolic alkalosis, achieved with dietary NaHCO3 or KHCO3 intake. Bicarbonate ion increases KCC3a abundance, both in vivo and in vitro, independently of the accompanying cation. Moreover, bicarbonate intake upregulates KCC3a independently of aldosterone or angiotensin II. Since NaHCO3 intake increased KCC3a abundance in wild-type as well as in pendrin knockout mice, this KCC3a upregulation by bicarbonate does not depend on a direct interaction with pendrin. We conclude that increased extracellular bicarbonate, as observed in models of metabolic alkalosis, directly raises KCC3a abundance independently of angiotensin II, aldosterone, or changes in KCC3a transcription and does not involve a direct interaction with pendrin.NEW & NOTEWORTHY KCC3a expression is stimulated in alkalemia. This paper shows that bicarbonate itself is mediating this effect through a posttranscriptional mechanism. The paper also shows that this phenomenon is not mediated by aldosterone or angiotensin II.

The role of Cl- and K+ efflux in NLRP3 inflammasome and innate immune response activation.

Inflammation is part of innate immunity and is a natural response of the body to bacteria, virus, and any other pathogen infections or to damaged tissues. However, too much inflammation or chronic inflammation contributes to a wide variety of diseases such as inflammatory bowel disease, cancer, type 2 diabetes, heart disease, and autoimmune diseases such as rheumatoid arthritis. Recent studies underscored the critical role of K+ and Cl- efflux in the activation of the inflammasome. The NLRP3 inflammasome is a multiprotein complex that mediates the production of the proinflammatory cytokines IL-1ß and IL-18 and initiates the inflammatory cell death or pyroptosis. The NLRP3 inflammasome can be activated by multiple stimuli such as extracellular ATP, microbial toxins, ROS, mitochondrial DNA, or particulate matter. Although the precise mechanisms of NLRP3 activation and regulation by these diverse agonists remain unclear, multiple reports indicate that all NLRP3 agonists ultimately lead to a drop in intracellular concentration of potassium (K+ efflux) and chloride (Cl- efflux). The WNK-SPAK/OSR1-[N]KCC pathway plays a critical role in maintaining K+ and Cl- ion concentrations in the cell. Recent advances indicate that the WNK-SPAK-[N]KCC pathway plays a role in the activation of the innate immune response. This review highlights recent discoveries detailing how ion transport regulates innate immune cell response to inflammatory stimuli.

Loss of NKCC1 function increases epithelial tight junction permeability by upregulating claudin-2 expression.

Conditions that cause the loss of epithelial barrier integrity are often accompanied by dysregulation of tight junction protein expression and/or localization. Recently, we have reported that patients with mutations in SLC12A2, the gene encoding the basolateral Na+-K+-2Cl- cotransporter (NKCC1), suffer from severe gastrointestinal deficits, including chronic gastrointestinal inflammation, gastrointestinal hemorrhage, intestinal obstruction, and constipation. Although the intestinal inflammation observed in patients with loss of NKCC1 function may or may not be due to tight junction dysfunction, we investigated whether the loss of NKCC1 function affects paracellular ion transport and epithelial barrier function. Wild-type HT29-MTX-E12 and CRISPR/Cas9-mediated NKCC1 knockout (KO) HT29 clones were tested for tight junction protein expression and localization. Tightness of epithelial cell monolayer was assessed by measurement of transepithelial electrical resistance and permeability of molecular tracers in transwell filters. Tight junction protein localization was assessed by immunofluorescence. Loss of NKCC1 expression strongly increases the expression of claudin-2 and occludin in epithelial cell monolayers. Loss of NKCC1 significantly reduces the transepithelial electrical resistance (TER) indicating an increase in paracellular ions flux, consistent with upregulation of the cation-selective and channel-forming claudin-2. In addition, NKCC1-KO monolayers showed a significant increase in the paracellular flux of small molecules like fluorescein (0.33 kDa), whereas the permeability of higher molecular weight TRITC-Dextran (4 kDa and 70 kDa) remained unchanged. Thus, NKCC1 regulates tight junction protein expression and loss of NKCC1 function affects epithelial barrier integrity.

Protein kinase D1 variant associated with human epilepsy and peripheral nerve hypermyelination.

We report the case of a patient with severe progressive epilepsy and peripheral neuropathy and a novel de novo inactivating variant (p.E79X) in Protein Kinase D1 (PKD1). Using CRISPR/Cas9, we engineered the homologous variant in mice and showed that in the homozygote mouse, it recapitulated the patient peripheral nerve hypermyelination pathology. The lethality of the homozygote mouse prevented us from performing an assessment of locomotor behavior. The mutant heterozygote mouse; however, exhibited a significant increase in kainate-induced seizure activity over wild-type mice, supporting the hypothesis that the PKD1 variant is a candidate for the cause of the patient epilepsy. Because PKD1 was previously identified in a kinomic screen as an interacting partner of the K-Cl cotransporter 3 (KCC3), and since KCC3 is involved in peripheral nerve disease and brain hyperexcitability, one possible mechanism of action of PKD1 in disease is through KCC3. We show that catalytically inactive PKD1 stimulates KCC3 activity, consistent with tonic relief of inhibitory phosphorylation. Our findings implicate a novel role for PKD1 in the human nervous system, and uncover a mechanism that could serve as a potential target to promote nervous system myelination.

A mutation in the Na-K-2Cl cotransporter-1 leads to changes in cellular metabolism.

The Na-K-Cl cotransporter-1 (NKCC1), by mediating the electroneutral transport of Na+ , K+ , and Cl- plays an important role in cell volume regulation, epithelial transport, and the control of neuronal excitability. Recently, we reported the first known human mutation in SLC12A2, the gene encoding NKCC1. The 17-year old patient suffers from multiorgan failure. Laboratory tests conducted on muscle and liver biopsies of the patient showed abnormal increase in mitochondrial DNA copy number and increased glycogen levels, indicating the possibility that the transporter may play a role in energy metabolism. Here, we show that fibroblasts isolated from the patient demonstrate a significant increase in mitochondrial respiration, compared to fibroblasts isolated from healthy individuals. Similarly, Madin Darby canine kidney (MDCK) cells transfected with enhanced green fluorescent protein (EGFP)-tagged mutant NKCC1 DNA demonstrated increased mitochondrial respiration when compared to MDCK cells expressing EGFP-tagged wild-type (WT) cotransporter. Direct inhibition of the cotransporter through addition of bumetanide did not change the rate of basal respiration, but led to increased maximal mitochondrial respiration. Fibroblasts extracted from NKCC1WT/DFX and NKCC1DFX/DFX mice also demonstrated a significant elevation in mitochondrial respiration, compared to fibroblasts isolated from their WT littermates. Expression of the mutant protein was associated with an increase in hydrogen peroxide and peroxidase activity and a decrease in messenger RNA transcript levels for protein involved in the unfolded protein response. These data reveal that cells expressing the mutant cotransporter demonstrate increased mitochondrial respiration and behave like they are experiencing a state of starvation.

A dileucine motif in the COOH-terminal domain of NKCC1 targets the cotransporter to the plasma membrane.

Na+-K+-2Cl- cotransporter-1 (NKCC1) mediates the electroneutral transport of Na+, K+, and Cl- and is normally localized to the basolateral membrane of polarized epithelial cells. We recently reported the first known solute carrier family 12 member 2 ( SLC12A2) mutation (we call NKCC1-DFX) that causes epithelial dysfunction in an undiagnosed disease program case. The heterozygous mutation leads to truncation of the COOH-terminal tail of the cotransporter, resulting in both mutant and wild-type cotransporters being mistrafficked to the apical membrane of polarized epithelial cells. Here we demonstrate by using consecutive truncations and site-directed mutagenesis of the COOH-terminal domain of NKCC1 that truncation of NKCC1 COOH domain uncouples the cotransporter from the lateral membrane. We identify a dileucine motif that, when mutated, leads to cotransporter accumulation in the cytoplasm and mistrafficking to the apical/subapical region of epithelial cells, thereby recapitulating the phenotype observed with the patient mutation. We show that truncation deletion and LL substitution mutants are trafficked out of the endoplasmic reticulum and trans-Golgi network but accumulate in early and late endosomes where they are degraded.

Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells.

We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na+-K+-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.

The Ste20 kinases SPAK and OSR1 travel between cells through exosomes.

Proteomics studies have identified Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) in exosomes isolated from body fluids such as blood, saliva, and urine. Because proteomics studies likely overestimate the number of exosome proteins, we sought to confirm and extend this observation using traditional biochemical and cell biology methods. We utilized HEK293 cells in culture to verify the packaging of these Ste20 kinases in exosomes. Using a series of centrifugation and filtration steps of conditioned culture medium isolated from HEK293 cells, we isolated nanovesicles in the range of 40-100 nm. We show that these small vesicles express the tetraspanin protein CD63 and lack endoplasmic reticulum and Golgi markers, consistent with these being exosomes. We show by Western blot and immunogold analyses that these exosomes express SPAK, OSR1, and Na-K-Cl cotransporter 1 (NKCC1). We show that exosomes are not only secreted by cells, but also accumulated by adjacent cells. Indeed, exposing cultured cells to exosomes produced by other cells expressing a fluorescently labeled kinase resulted in the kinase finding its way into the cytoplasm of these cells, consistent with the idea of exosomes serving as cell-to-cell communication vessels. Similarly, coculturing cells expressing different fluorescently tagged proteins resulted in the exchange of proteins between cells. In addition, we show that both SPAK and OSR1 kinases entering cells through exosomes are preferentially expressed at the plasma membrane and that the kinases in exosomes are functional and maintain NKCC1 in a phosphorylated state.

TGFß loss activates ADAMTS-1-mediated EGF-dependent invasion in a model of esophageal cell invasion.

The TGFß signaling pathway is essential to epithelial homeostasis and is often inhibited during progression of esophageal squamous cell carcinoma. Recently, an important role for TGFß signaling has been described in the crosstalk between epithelial and stromal cells regulating squamous tumor cell invasion in mouse models of head-and-neck squamous cell carcinoma (HNSCC). Loss of TGFß signaling, in either compartment, leads to HNSCC however, the mechanisms involved are not well understood. Using organotypic reconstruct cultures (OTC) to model the interaction between epithelial and stromal cells that occur in dysplastic lesions, we show that loss of TGFß signaling promotes an invasive phenotype in both fibroblast and epithelial compartments. Employing immortalized esophageal keratinocytes established to reproduce common mutations of esophageal squamous cell carcinoma, we show that treatment of OTC with inhibitors of TGFß signaling (A83-01 or SB431542) enhances invasion of epithelial cells into a fibroblast-embedded Matrigel/collagen I matrix. Invasion induced by A83-01 is independent of proliferation but relies on protease activity and expression of ADAMTS-1 and can be altered by matrix density. This invasion was associated with increased expression of pro-inflammatory cytokines, IL1 and EGFR ligands HB-EGF and TGFα. Altering EGF signaling prevented or induced epithelial cell invasion in this model. Loss of expression of the TGFß target gene ROBO1 suggested that chemorepulsion may regulate keratinocyte invasion. Taken together, our data show increased invasion through inhibition of TGFß signaling altered epithelial-fibroblasts interactions, repressing markers of activated fibroblasts, and altering integrin-fibronectin interactions. These results suggest that inhibition of TGFß signaling modulates an array of pathways that combined promote multiple aspects of tumor invasion.

SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells.

BACKGROUND: Tumor metastasis is responsible for 90% of cancer-related deaths. Recently, a strong link between microRNA dysregulation and human cancers has been established. However, the molecular mechanisms through which microRNAs regulate metastasis and cancer progression remain unclear. METHODS: We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western blotting using overexpression and shRNA knock-down approaches. Furthermore, methylation studies were used to assess epigenetic regulation of expression. Functionally, we determined the cellular consequences using migration and invasion assays, as well as proliferation assays. Immunoprecipitation and ChIP were used to identify complex formation of SOX4 and co-repressor components. RESULTS: Here, we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues. CONCLUSIONS: Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

Fetuin-A associates with histones intracellularly and shuttles them to exosomes to promote focal adhesion assembly resulting in rapid adhesion and spreading in breast carcinoma cells.

The present analyses were undertaken to define the mechanisms by which fetuin-A modulates cellular adhesion. FLAG-tagged fetuin-A was expressed in breast carcinoma and HEK-293T cells. We demonstrated by confocal microscopy that fetuin-A co-localizes with histone H2A in the cell nucleus, forms stable complexes with histones such as H2A and H3 in solution, and shuttles histones to exosomes. The rate of cellular adhesion and spreading to either fibronectin or laminin coated wells was accelerated significantly in the presence of either endogenous fetuin-A or serum derived protein. More importantly, the formation of focal adhesion complexes on surfaces coated by laminin or fibronectin was accelerated in the presence of fetuin-A or histone coated exosomes. Cellular adhesion mediated by histone coated exosomes was abrogated by heparin and heparinase III. Heparinase III cleaves heparan sulfate from cell surface heparan sulfate proteoglycans. Lastly, the uptake of histone coated exosomes and subsequent cellular adhesion, was abrogated by heparin. Taken together, the data suggest a mechanism where fetuin-A, either endogenously synthesized or supplied extracellularly can extract histones from the nucleus or elsewhere in the cytosol/membrane and load them on cellular exosomes which then mediate adhesion by interacting with cell surface heparan sulfate proteoglycans via bound histones.

Alpha-2 Heremans Schmid Glycoprotein (AHSG) modulates signaling pathways in head and neck squamous cell carcinoma cell line SQ20B.

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-ß was examined to determine whether levels of the TGF-ß binding AHSG influenced the effect of TGF-ß on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-ß influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis.

Kinase Scaffold Cab39 Is Necessary for Phospho-Activation of the Thiazide-Sensitive NCC.

BACKGROUND: Potassium regulates the WNK (with no lysine kinase)-SPAK (STE20/SPS1-related proline/alanine-rich kinase) signaling axis, which in turn controls the phosphorylation and activation of the distal convoluted tubule thiazide-sensitive NCC (sodium-chloride cotransporter) for sodium-potassium balance. Although their roles in the kidney have not been investigated, it has been postulated that Cab39 (calcium-binding protein 39) or Cab39l (Cab39-like) is required for SPAK/OSR1 (oxidative stress response 1) activation. This study demonstrates how they control the WNK-SPAK/OSR1-NCC pathway. METHODS: We created a global knockout of Cab39l and a tamoxifen-inducible, NCC-driven, Cab39 knockout. The 2 lines were crossed to generate Cab39-DKO (Cab39 double knockout) animals. Mice were studied under control and low-potassium diet, which activates WNK-SPAK/OSR1-NCC phosphorylation. Western blots were used to assess the expression and phosphorylation of proteins. Blood and urine electrolytes were measured to test for compromised NCC function. Immunofluorescence studies were conducted to localize SPAK and OSR1. RESULTS: Both Cab39l and Cab39 are expressed in distal convoluted tubule, and only the elimination of both leads to a striking absence of NCC phosphorylation. Cab39-DKO mice exhibited a loss-of-NCC function, like in Gitelman syndrome. In contrast to the apical membrane colocalization of SPAK with NCC in wild-type mice, SPAK and OSR1 become confined to intracellular puncta in the Cab39-DKO mice. CONCLUSIONS: In the absence of Cab39 proteins, NCC cannot be phosphorylated, resulting in a Gitelman-like phenotype. Cab39 proteins function to localize SPAK at the apical membrane with NCC, reminiscent of the Cab39 yeast homolog function, translocating kinases during cytokinesis.

Reduced annexin A6 expression promotes the degradation of activated epidermal growth factor receptor and sensitizes invasive breast cancer cells to EGFR-targeted tyrosine kinase inhibitors.

BACKGROUND: The expression of annexin A6 (AnxA6) in AnxA6-deficient non-invasive tumor cells has been shown to terminate epidermal growth factor receptor (EGFR) activation and downstream signaling. However, as a scaffolding protein, AnxA6 may stabilize activated cell-surface receptors to promote cellular processes such as tumor cell motility and invasiveness. In this study, we investigated the contribution of AnxA6 in the activity of EGFR in invasive breast cancer cells and examined whether the expression status of AnxA6 influences the response of these cells to EGFR-targeted tyrosine kinase inhibitors (TKIs) and/or patient survival. RESULTS: We demonstrate that in invasive BT-549 breast cancer cells AnxA6 expression is required for sustained membrane localization of activated (phosho-Y1068) EGFR and consequently, persistent activation of MAP kinase ERK1/2 and phosphoinositide 3-kinase/Akt pathways. Depletion of AnxA6 in these cells was accompanied by rapid degradation of activated EGFR, attenuated downstream signaling and as expected enhanced anchorage-independent growth. Besides inhibition of cell motility and invasiveness, AnxA6-depleted cells were also more sensitive to the EGFR-targeted TKIs lapatinib and PD153035. We also provide evidence suggesting that reduced AnxA6 expression is associated with a better relapse-free survival but poorer distant metastasis-free and overall survival of basal-like breast cancer patients. CONCLUSIONS: Together this demonstrates that the rapid degradation of activated EGFR in AnxA6-depleted invasive tumor cells underlies their sensitivity to EGFR-targeted TKIs and reduced motility. These data also suggest that AnxA6 expression status may be useful for the prediction of the survival and likelihood of basal-like breast cancer patients to respond to EGFR-targeted therapies.

Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions.

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell-cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell-cell and/or cell-ECM contacts and anchorage-independent cell proliferation.

KCC3a, a Strong Candidate Pathway for K+ Loss in Alkalemia.

Loss-of-function mutations in the human potassium chloride cotransporter-3 (KCC3) cause a hereditary motor sensory neuropathy associated with agenesis of the corpus callosum. While recapitulating the neuropathy, KCC3-knockout mice also exhibit high blood pressure. This phenotype is believed to have neurogenic and/or vascular origins. The role of KCC3 in the kidney is poorly understood. KCC3 is encoded by two major isoforms originating from alternative promoters: KCC3a and KCC3b, with KCC3b being the predominant transcript in the kidney. Although the transporter has previously been localized to the proximal tubule, we show here the unique expression of the KCC3a isoform in the connecting tubule. Using a KCC3a-specific polyclonal antibody validated for both immunofluorescence and immunoblotting, we showed an intense KCC3a signal restricted to cortical intercalated cells. No overlap is detected between KCC3a and sodium chloride cotransporter (NCC), a distal convoluted tubule (DCT) marker; or between KCC3a and ENaC or calbindin, which are both principal cell markers. KCC3a signal was observed in cells expressing the apical V-ATPase and pendrin, establishing a unique expression pattern characteristic of intercalated cells of type-B or type-nonA/nonB. We further show that treatment of wild-type mice with hydrochlorothiazide, amiloride, or fed a K+-deficient diet up-regulates KCC3a level, suggesting that volume depletion increases KCC3a abundance. This hypothesis was confirmed by showing a higher abundance of KCC3a protein after 23-h water restriction or after placing the mice on a low-salt diet. More importantly, abundance of the Cl-/HCO3 - exchanger, pendrin, which is known to secrete bicarbonate in alkalotic conditions, was significantly diminished in KCC3-knockout mice. In addition, KCC3a abundance increased significantly alongside pendrin abundance in bicarbonate-treated alkalotic mice, providing a credible mechanism for K+ loss in metabolic alkalosis.

Fetuin-A ({alpha}2HS-glycoprotein) is a major serum adhesive protein that mediates growth signaling in breast tumor cells.

The identity of the cell adhesive factors in fetal bovine serum, commonly used to supplement growth media, remains a mystery due to the plethora of serum proteins. In the present analyses, we showed that fetuin-A, whose function in cellular attachment in tissue culture has been debated for many years, is indeed a major serum cell attachment factor particularly for tumor cells. We are able to report this because of a new purification strategy that has for the first time given us a homogeneous protein band in colloidal Coomassie-stained gels that retains biological activity. The tumor cells adhered to immobilized fetuin-A and not α(2)-macroglobulin, its major contaminant. The interaction of cells with fetuin-A was driven mainly by Ca(2+) ions, and cells growing in regular medium supplemented with fetal bovine serum were just as sensitive to loss of extracellular Ca(2+) ions as cells growing in fetuin-A. Fractionation of human serum revealed that cell attachment was confined to the fractions that had fetuin-A. Interestingly, the tumor cells also took up fetuin-A and secreted it back to the medium using an unknown mechanism that can be observed in live cells. The attachment of tumor cells to fetuin-A was accompanied by phosphatidylinositol 3-kinase/Akt activation that was down-regulated in cells that lack annexin-A6, one of the cell surface receptors for fetuin-A. Taken together, our data show the significance of fetuin-A in tumor cell growth mechanisms in vitro and open new research vistas for this protein.

Lack of fetuin-A (alpha2-HS-glycoprotein) reduces mammary tumor incidence and prolongs tumor latency via the transforming growth factor-beta signaling pathway in a mouse model of breast cancer.

The present analyses were done to define the role of fetuin-A (Fet) in mammary tumorigenesis using the polyoma middle T antigen (PyMT) transgenic mouse model. We crossed Fet-null mice in the C57BL/6 background with PyMT mice in the same background and after a controlled breeding protocol obtained PyMT/Fet+/+, PyMT/Fet+/-, and PyMT/Fet-/- mice that were placed in control and experimental groups. Whereas the control group (PyMT/Fet+/+) formed mammary tumors 90 days after birth, tumor latency was prolonged in the PyMT/Fet-/- and PyMT/Fet+/- mice. The majority of the PyMT/Fet-/- mice were tumor-free at the end of the study, at approximately 40 weeks. The pathology of the mammary tumors in the Fet-null mice showed extensive fibrosis, necrosis, and squamous metaplasia. The preneoplastic mammary tissues of the PyMT/Fet-/- mice showed intense phopho-Smad2/3 staining relative to control tissues, indicating that transforming growth factor-ß signaling is enhanced in these tissues in the absence of Fet. Likewise, p19ARF and p53 were highly expressed in tumor tissues of PyMT/Fet-/- mice relative to the controls in the absence of Fet. The phosphatidylinositol 3-kinase/Akt signaling pathway that we previously showed to be activated by Fet, on the other hand, was unaffected by the absence of Fet. The data indicate that Fet is a powerful modulator of breast tumorigenesis in this model system and has the potential to modulate breast cancer progression in humans.