Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts.
ABSTRACT The Cl/HCO(3) exchanger downregulated in adenoma (DRA) mediates electroneutral NaCl absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity.
- SourceAvailable from: Jaleh Malakooti[show abstract] [hide abstract]
ABSTRACT: The epithelial apical membrane Na+/H+ exchangers [NHE (sodium hydrogen exchanger)2 and NHE3] and Cl-/HCO3- exchangers [DRA (down-regulated in adenoma) and PAT-1 (putative anion transporter 1)] are key luminal membrane transporters involved in electroneutral NaCl absorption in the mammalian intestine. During the last decade, there has been a surge of studies focusing on the short-term regulation of these electrolyte transporters, particularly for NHE3 regulation. However, the long-term regulation of the electrolyte transporters, involving transcriptional mechanisms and transcription factors that govern their basal regulation or dysregulation in diseased states, has only now started to unfold with the cloning and characterization of their gene promoters. The present review provides a detailed analysis of the core promoters of NHE2, NHE3, DRA and PAT-1 and outlines the transcription factors involved in their basal regulation as well as in response to both physiological (butyrate, protein kinases and probiotics) and pathophysiological (cytokines and high levels of serotonin) stimuli. The information available on the transcriptional regulation of the recently identified NHE8 isoform is also highlighted. Therefore the present review bridges a gap in our knowledge of the transcriptional mechanisms underlying the alterations in the gene expression of intestinal epithelial luminal membrane Na+ and Cl- transporters involved in electroneutral NaCl absorption. An understanding of the mechanisms of the modulation of gene expression of these transporters is important for a better assessment of the pathophysiology of diarrhoea associated with inflammatory and infectious diseases and may aid in designing better management protocols.Biochemical Journal 04/2011; 435(2):313-25. · 4.65 Impact Factor
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ABSTRACT: Sperm capacitation is required for fertilization and involves several ion permeability changes. Although Cl(-) and HCO(3)(-) are essential for capacitation, the molecular entities responsible for their transport are not fully known. During mouse sperm capacitation, the intracellular concentration of Cl(-) ([Cl(-)](i)) increases and membrane potential (Em) hyperpolarizes. As in noncapacitated sperm, the Cl(-) equilibrium potential appears to be close to the cell resting Em, opening of Cl(-) channels could not support the [Cl(-)](i) increase observed during capacitation. Alternatively, the [Cl(-)](i) increase might be mediated by anion exchangers. Among them, SLC26A3 and SLC26A6 are good candidates, since, in several cell types, they increase [Cl(-)](i) and interact with cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel present in mouse and human sperm. This interaction is known to be mediated and probably regulated by the Na(+)/H(+) regulatory factor-1 (official symbol, SLC9A3R1). Our RT-PCR, immunocytochemistry, Western blot, and immunoprecipitation data indicate that SLC26A3, SLC26A6, and SLC9A3R1 are expressed in mouse sperm, localize to the midpiece, and interact between each other and with CFTR. Moreover, we present evidence indicating that CFTR and SLC26A3 are involved in the [Cl(-)](i) increase induced by db-cAMP in noncapacitated sperm. Furthermore, we found that inhibitors of SLC26A3 (Tenidap and 5099) interfere with the Em changes that accompany capacitation. Together, these findings indicate that a CFTR/SLC26A3 functional interaction is important for mouse sperm capacitation.Biology of Reproduction 01/2012; 86(1):1-14. · 4.03 Impact Factor
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ABSTRACT: Response rates to chemotherapy remain highly variable in breast cancer patients. We set out to identify genes associated with chemotherapy resistance. We analyzed what is currently the largest single-institute set of gene expression profiles derived from breast cancers prior to a single neoadjuvant chemotherapy regimen (dose-dense doxorubicin and cyclophosphamide). We collected, gene expression-profiled, and analyzed 178 HER2-negative breast tumor biopsies ("NKI dataset"). We employed a recently developed approach for detecting imbalanced differential signal (DIDS) to identify markers of resistance to treatment. In contrast to traditional methods, DIDS is able to identify markers that show aberrant expression in only a small subgroup of the non-responder samples. We found a number of markers of resistance to anthracycline-based chemotherapy. We validated our findings in three external datasets, totaling 456 HER2-negative samples. Since these external sets included patients who received differing treatment regimens, the validated markers represent markers of general chemotherapy resistance. There was a highly significant overlap in the markers identified in the NKI dataset and the other three datasets. Five resistance markers, SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B, were identified in three of the four datasets (p value overlap < 1 × 10(-6)). These five genes identified resistant tumors that could not have been identified by merely taking ER status or proliferation into account. The identification of these genes might lead to a better understanding of the mechanisms involved in (clinically) observed chemotherapy resistance and could possibly assist in the recognition of breast cancers in which chemotherapy does not contribute to response or survival.Breast Cancer Research and Treatment 12/2012; · 4.47 Impact Factor