Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b^0,+AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b^0,+AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.The heteromeric amino acid transporter (HAT) family is one of the major amino acid transporter families responsible for cellular uptake and epithelial transport (1–3). HATs form heterodimers composed of a 12 membrane spanning light chain (SLC7) that catalyzes transport functions and a single membrane spanning heavy chain (SLC3) essential for plasma membrane localization and stabilization of the light chains. Two heavy chains, SLC3A1/rBAT and SLC3A2/4F2hc/CD98hc, covalently bound to light chains via a disulfide bridge have been identified so far (4–6). 4F2hc interacts with most of the light chains in HATs whereas rBAT has been known to form a heterodimer only with b^0,+AT/SLC7A9. Because the rBAT-b^0,+AT complex is presented on the apical membrane of proximal tubules in the kidney and involved in the reabsorption of cystine and dibasic amino acids, the mutations of either rBAT or b^0,+AT cause cystinuria, a disorder of renal reabsorption of cystine and dibasic amino acids leading to serious renal lithiasis due to low solubility of cystine (7).An unsolved paradox on rBAT and b^0,+AT has been the discrepancy between the distribution of rBAT and that of b^0,+AT (5, 8–10). rBAT is the most abundant in the S3 segment of proximal tubules, and its expression declines toward the S1 segment (11, 12). In contrast, the expression of b^0,+AT is highest in the S1 segment and decreases toward the S3 segment (5, 8). Furthermore, even in b^0,+AT-deficient mice, heterodimers containing rBAT still have been observed (13). Therefore, it has been proposed that unknown partners of rBAT exist in the S3 segment (5, 9, 14, 15).The HAT family includes two members, AGT1/SLC7A13 and Asc2, whose heavy chains have not been identified (16, 17). Among them, aspartate/glutamate transporter 1 (AGT1) has been identified as an Na⁺-independent acidic amino acid transporter expressed specifically in the kidney (17). In this study, we have generated new anti-AGT1 antibodies to search for the unknown heavy chain(s), by means of coimmunoprecipitation followed by mass spectrometry, and have revealed that rBAT is a heavy chain of AGT1. AGT1 was detected at the apical membrane of the S3 segment in renal proximal tubules. A transport assay of the AGT1-rBAT heterodimer reconstituted into proteoliposomes revealed that it transports cystine as well as aspartate and glutamate. We conclude that AGT1 is a strong candidate for the “missing partner” of rBAT and a second cystine transporter in the kidney.     

Case report of a laparoscopically resected rectal villous tumor associated with electrolyte depletion syndrome

A 70‐year‐old woman had been aware of lower extremity weakness and anal discomfort for 3 years. A soft, elastic, palm‐sized mass covered by a large amount of mucus was found protruding from the anus. Biopsy revealed a villous adenoma. On the basis of these results, a villous adenoma associated with electrolyte depletion syndrome was diagnosed. After electrolyte abnormalities were improved by fluid replacement therapy, laparoscopic abdominoperineal resection was performed. The surgically resected specimen was a circumferential villous tumor measuring 210 × 140 mm. The histopathological diagnosis was an intramucosal papillary adenocarcinoma. The patient recovered uneventfully after surgery, and the electrolyte abnormalities gradually improved. She was discharged on the 28th postoperative day. The electrolyte levels normalized about 3 months after surgery.     

Papillary Fibroelastoma of the Aortic Valve: Analysis of 21 Cases,Including a Presentation with Cardiac Arrest

Cardiac papillary fibroelastoma is a rare, benign tumor, arising predominantly from cardiac valves. This tumor can cause a variety of symptoms due to thromboembolism. We describe our single-center surgical experience with papillary fibroelastoma of the aortic valve.From April 2004 through June 2013, 6,530 patients underwent cardiac surgery. Of those, 6,098 patients were included in the final analysis. Twenty-one patients (0.34%) underwent surgical resection of 30 papillary fibroelastomas of the aortic valve.Most patients (67%) were incidentally diagnosed to have cardiac papillary fibroelastoma. The usual symptom was cerebral infarction (in 5 of 7 symptomatic patients). A rare presentation of papillary fibroelastoma in one patient was cardiac arrest caused by left main coronary artery ostial obstruction. Tumor size was not related to patient age (Pearson correlation coefficient, 0.34; P=0.13). Neither the number of tumors (1.43 ± 0.72 vs 1.43 ± 0.62) nor tumor size (8.14 ± 2.42 vs 8.07 ± 3.31 mm) was significantly different between symptomatic and asymptomatic patients. All lesions were resected by means of the simple shave technique. There were no operative or 30-day deaths. Follow-up echocardiograms showed no tumor recurrence (mean follow-up duration, 17 ± 14 mo).We identified no significant relationship among tumor size, number of tumors, symptoms, or patient age. Because simple shave excision of the tumor can be safely achieved without evidence of tumor recurrence, we conclude that surgical resection can be reasonable in asymptomatic patients.     

Growth factors change nuclear distribution of estrogen receptor-alpha via mitogen-activated protein kinase or phosphatidylinositol 3-kinase cascade in a human breast cancer cell line

In the present study, to examine the dynamic changes in the localization of nuclear estrogen receptor (ER)alpha induced by growth factors, we used time-lapse confocal microscopy to directly visualized ERalpha fused with green fluorescent protein (GFP-ERalpha) in single living cells treated with epidermal growth factor (EGF) or IGF-I. We observed that 17beta-estradiol (E2) changed the normally diffuse distribution of GFP-ERalpha throughout the nucleoplasm to a hyperspeckled distribution within 10 min. Both EGF and IGF-I also changed the nuclear distribution of GFP-ERalpha, similarly to E2 treatment. However, the time courses of the nuclear redistribution of GFP-ERalpha induced by EGF or IGF-I were different from that induced by E2 treatment. In the EGF-treated cells, the GFP-ERalpha nuclear redistribution was observed at 30 min and reached a maximum at 60 min, whereas in the IGF-I-treated cells, the GFP-ERalpha nuclear redistribution was observed at 60 min and reached a maximum at 90 min. The EGF-induced redistribution of GFP-ERalpha was blocked by pretreatment with a MAPK cascade inhibitor, PD98059, whereas the IGF-I-induced redistribution of GFP-ERalpha was blocked by pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002. Analysis using an activation function-2 domain deletion mutant of GFP-ERalpha showed that the change in the distribution of GFP-ERalpha was not induced by E2, EGF, or IGF-I treatment. These data suggest that MAPK and phosphatidylinositol 3-kinase cascades are involved in the nuclear redistribution of ERalpha by EGF and IGF-I, respectively, and that the activation function-2 domain of ERalpha may be needed for the nuclear redistribution of ERalpha.     

Interferon-alphaCon1 suppresses proliferation of liver cancer cell lines in vitro and in vivo

BACKGROUND/AIMS: We investigated the effects of consensus interferon (IFN-alphaCon1), a nonnaturally occurring type I interferon with higher specific activity than other type I IFNs, on the growth of human liver cancer cells. METHODS: The effect of IFN-alphaCon1 on the proliferation of 13 liver cancer cell lines was investigated in vitro. Hepatocellular carcinoma (HCC) cells (KIM-1 and HAK-1B) were transplanted subcutaneously into the back of nude mice, then IFN-alphaCon1 was subcutaneously administered to the mice once a day for 2 weeks, and tumor volume and histology were examined. RESULTS: IFN-alphaCon1 expressed a dose-dependent growth inhibitory effect in all cell lines in vitro. KIM-1 tumor volume in mice that received 0.01 microg (10(4)IU)/mouse/day of IFN-alphaCon1 (similar to the clinical dose for chronic hepatitis C) was 62% of the control, 0.1microg/mouse/day resulted in 26%, and 1 microg/mouse/day resulted in 10%. HAK-1B tumor volume under the same treatment was 61, 24 and 0% of the control, respectively. The number of apoptotic cells significantly increased and the number of blood vessels significantly decreased with the increase in IFN-alphaCon1 dose. CONCLUSIONS: IFN-alphaCon1 suppressed HCC growth in nude mice. These data indicate the potential clinical application of IFN-alphaCon1 in the prevention and treatment of HCC.     

Enhancer of zeste homolog 2 (EZH2) regulates tumor angiogenesis and predicts recurrence and prognosis of intrahepatic cholangiocarcinoma

Background

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and regulates tumor malignancy by gene silencing via histone methylation. In this study we investigate the role of EZH2 in angiogenesis of intrahepatic cholangiocarcinoma (ICC).

Gastrin receptor genes are expressed in gastric parietal and enterochromaffin-like cells ofMastomys natalensis

Although gastric enterochromaffin-like (ECL) carcinoid tumors are known to develop in patients with long-standing hypergastrinemia, the expression of the gastrin receptor gene in ECL cells has not yet been demonstrated. Therefore, this study was designed to examine gastrin receptor gene expression in ECL cells.Mastomys gastric mucosal cells isolated by enzyme dispersion were separated into 10 fractions (F1–10) by centrifugal elutriation. Each fraction was examined histologically to determine whether they contained ECL and/or parietal cells and Northern blot analysis was used to confirm the presence of histidine decarboxylase and H⁺, K⁺-ATPase gene expression. ECL cells were found only in fractions 1 and 2, whereas parietal cells were detected in fractions 6–10. Gastrin receptor gene expression was demonstrated in both parietal cell-rich and ECL cell-rich fractions. In addition, the gastrin receptor cDNA sequences obtained from the two of the fractions (F1 and 8) were identical. These results suggest that gastrin receptor genes are expressed in ECL cells as well as in parietal cells and that these receptors are identical.This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture, and a grant from the Ministry of Health and Welfare, Japan.