Both IRBIT and long-IRBIT bind to and coordinately regulate Cl−/HCO3− exchanger AE2 activity through modulating the lysosomal degradation of AE2

Anion exchanger 2 (AE2) plays crucial roles in regulating cell volume homeostasis and cell migration. We found that both IRBIT and Long-IRBIT (L-IRBIT) interact with anion exchanger 2 (AE2). The interaction occurred between the conserved AHCY-homologous domain of IRBIT/L-IRBIT and the N-terminal cytoplasmic region of AE2. Interestingly, AE2 activity was reduced in L-IRBIT KO cells, but not in IRBIT KO cells. Moreover, AE2 activity was slightly increased in IRBIT/L-IRBIT double KO cells. These changes in AE2 activity resulted from changes in the AE2 expression level of each mutant cell, and affected the regulatory volume increase and cell migration. The activity and expression level of AE2 in IRBIT/L-IRBIT double KO cells were downregulated if IRBIT, but not L-IRBIT, was expressed again in the cells, and the downregulation was cancelled by the co-expression of L-IRBIT. The mRNA levels of AE2 in each KO cell did not change, and the downregulation of AE2 in L-IRBIT KO cells was inhibited by bafilomycin A1. These results indicate that IRBIT binding facilitates the lysosomal degradation of AE2, which is inhibited by coexisting L-IRBIT, suggesting a novel regulatory mode of AE2 activity through the binding of two homologous proteins with opposing functions.

Both Irbit and Long-Irbit Bind to and Coordinately Regulate Cl-/hco3-Exchanger Ae2 Activity Through Modulating the Lysosomal Degradation of Ae2.

Anion exchanger 2 (AE2) plays crucial roles in regulating cell volume homeostasis and cell migration. We found that both IRBIT and Long-IRBIT (L-IRBIT) interact with anion exchanger 2 (AE2). The interaction occurred between the conserved AHCY-homologous domain of IRBIT/L-IRBIT and the N-terminal cytoplasmic region of AE2. Interestingly, AE2 activity was reduced in L-IRIBT KO cells, but not in IRBIT KO cells. Moreover, AE2 activity was slightly increased in IRBIT/L-IRBIT double KO cells. These changes in AE2 activity resulted from changes in the AE2 expression level of each mutant cell, and affected the regulatory volume increase and cell migration. The activity and expression level of AE2 in IRBIT/L-IRBIT double KO cells were downregulated if IRBIT, but not LIRBIT, was expressed again in the cells, and the downregulation was cancelled by the co-expression of L-IRBIT. The mRNA levels of AE2 in each KO cell did not change, and the downregulation of AE2 in L-IRBIT KO cells was inhibited by bafilomycin A1. These results indicate that IRBIT binding facilitates the lysosomal degradation of AE2, which is inhibited by coexisting L-IRBIT, suggesting a novel regulatory mode of AE2 activity through the binding of two homologous proteins with opposing functions.

Drug Repurposing as an Antitumor Agent: Disulfiram-Mediated Carbonic Anhydrase 12 and Anion Exchanger 2 Modulation to Inhibit Cancer Cell Migration

Disulfiram has been used in the treatment of alcoholism and exhibits an anti-tumor effect. However, the intracellular mechanism of anti-tumor activity of Disulfiram remains unclear. In this study, we focused on the modulatory role of Disulfiram via oncogenic factor carbonic anhydrase CA12 and its associated transporter anion exchanger AE2 in lung cancer cell line A549. The surface expression of CA12 and AE2 were decreased by Disulfiram treatment with a time-dependent manner. Disulfiram treatment did not alter the expression of Na+-bicarbonate cotransporters, nor did it affect autophagy regulation. The chloride bicarbonate exchanger activity of A549 cells was reduced by Disulfiram treatment in a time-dependent manner without change in the resting pH level. The expression and activity of AE2 and the expression of CA12 were also reduced by Disulfiram treatment in the breast cancer cell line. An invasion assay and cell migration assay revealed that Disulfiram attenuated the invasion and migration of A549 cells. In conclusion, the attenuation of AE2 and its supportive enzyme CA12, and the inhibitory effect on cell migration by Disulfiram treatment in cancer cells provided the molecular evidence supporting the potential of Disulfiram as an anticancer agent.

Drosophila anion exchanger 2 is required for proper ovary development and oogenesis

Understanding how cell fate decisions are regulated is a central question in stem cell biology. Recent studies have demonstrated that intracellular pH (pHi) dynamics contribute to this process. Indeed, the pHi of cells within a tissue is not simply a consequence of chemical reactions in the cytoplasm and other cellular activity, but is actively maintained at a specific setpoint in each cell type. We found previously that the pHi of cells in the follicle stem cell (FSC) lineage in the Drosophila ovary increases progressively during differentiation from an average of 6.8 in the FSCs, to 7.0 in newly produced daughter cells, to 7.3 in more differentiated cells. Two major regulators of pHi in this lineage are Drosophila sodium-proton exchanger 2 (dNhe2) and a previously uncharacterized gene, CG8177, that is homologous to mammalian anion exchanger 2 (AE2). Based on this homology, we named the gene ae2. Here, we generated null alleles of ae2 and found that homozygous mutant flies are viable but have severe defects in ovary development and adult oogenesis. Specifically, we find that ae2 null flies have smaller ovaries, reduced fertility, and impaired follicle formation. In addition, we find that the follicle formation defect can be suppressed by a decrease in dNhe2 copy number and enhanced by the overexpression of dNhe2, suggesting that this phenotype is due to the dysregulation of pHi. These findings support the emerging idea that pHi dynamics regulate cell fate decisions and our studies provide new genetic tools to investigate the mechanisms by which this occurs.

PS02.188: EXPRESSION AND ROLE OF ANION EXCHANGER 2 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Background Purpose: Recent studies have reported essential roles for various intracellular pH regulators in epithelial carcinogenesis and tumor progression. The aims of the present study were to investigate the role of anion exchanger 2 (AE2) in the regulation of tumor progression-related genes and the prognostic value of its expression in esophageal squamous cell carcinoma (ESCC). Methods In human ESCC cell lines, knockdown experiments were conducted using AE2 siRNA, and the effects on cellular movement and survival were analyzed. The gene expression profiles of cells were examined using a microarray analysis. An immunohistochemical analysis was performed on 61 primary tumor samples obtained from ESCC patients who underwent esophagectomy. Results AE2 was strongly expressed in KYSE170 and TE13 cells. The depletion of AE2 in these cells increased cell migration and inhibited the induction of apoptosis. The results of the microarray analysis revealed that various matrix metalloproteinase (MMP) signaling pathway-related genes, such as MMP1, MMP12, and TIMP4, were up- or down-regulated in AE2-depleted KYSE170 cells. Immunohistochemical staining showed that AE2 was primarily located in the cell membranes or cytoplasm of carcinoma cells, and its expression pattern at the invasive front (IF) of the tumor was related to the pT category. Prognostic analyses revealed that the low-grade expression of AE2 at the IF was associated with shorter postoperative survival. Conclusion s: The results of the present study suggest that reductions in AE2 in ESCC enhance cellular movement by affecting MMP signaling pathways and are related to a poor prognosis in patients with ESCC. Disclosure All authors have declared no conflicts of interest.