Tomoko Nii

Human L-type amino acid transporter 1 (LAT1) : characterization of function and expression in tumor cell lines

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.

Dietary regulation of renal phosphate transporters in hypophosphatemic mice

Abstract: X-linked hypophosphatemic rickets (XLH) is known to impair renal adaptive response to Pi restriction. We investigated the effects of dietary Pi on the synthesis of renal sodium-dependent inorganic phosphate (Na/Pi) cotransporters (Npt1 and NaPi-7) in X-linked hypophosphatemic mice (Hyp). The NaPi-7 mRNA level in Hyp mice was reduced to 50% of that of normal mice while the Npt1 mRNA level was unchanged. After feeding a low-Pi diet, the amounts of NaPi-7 protein and mRNA were markedly increased in both normal and Hyp mice. In contrast, after feeding a high-Pi diet, the levels of protein and mRNA were largely decreased in both mice. Immunohistochemical analysis indicated that NaPi-7 staining was largely enhanced in the apical membrane of renal proximal tubular cells in the normal and Hyp mice fed the low-Pi diet. In contrast, NaPi-7 staining was decreased in both groups of rats fed the high-Pi diet. Npt1 immunoreactivity was detected in the apical membrane of proximal convoluted and straight tubular cells in Hyp and normal mice, and was unchanged regardless of dietary Pi manipulation in both mice. Thus, dietary regulation for the synthesis of the two cotransporters is not impaired in Hyp mice.

Direct demonstration of humorally mediated inhibition of the transcription of phosphate transporter in XLH patients

AbstractBackground. X-linked hypophosphatemia (XLH) is the most common form of familial hypophosphatemic rickets, which results from impaired renal phosphate (Pi) reabsorption, renal vitamin D metabolism, and skeletal mineralization. PHEX the gene responsible for XLH, is homologous to members of the neutral endopeptidase family. The role that the PHEX gene plays in Pi transport is not yet known. Methods. In the present study, we investigated the molecular mechanisms of the disorder of renal Pi transport in XLH in seven individuals from Japanese XLH families. Also, to characterize the effect of XLH serum from these patients on the expression of a renal phosphate transporter gene, we performed a functional analysis of the human type II Na/Pi cotransporter (NPT2) gene promoter in a kidney cell line, OK cells (named OK-B2400), which had stable expression of the luciferase reporter vector p3P2400 with 2462 base pairs (nucleotides −2409 to +53) of the NPT2 gene. Results. XLH serum significantly decreased the transcriptional activity of the NPT2 promoter. The mutations identified in our patients included three large deletions, frame shift mutations, and a stop mutation in the PHEX gene, which suggest that the gene is nonfunctional in affected individuals. Conclusion. The present study demonstrated that the serum of XLH patients with PHEX mutations contains inhibitory factor(s) for the transcriptional activity of the NPT2 gene. In addition, the transcription assay of the NPT2 promoter may provide a useful tool for the characterization of the putative PHEX protein substrate.

Molecular and cellular regulation of renal phosphate transporters in X-linked hypophosphatemia

ConclusionRecent investigations of X-linked hypophosphatemia support the concept that the kidney is intrinsically normal in this disorder, and that the characteristic phosphaturia is caused by a humoral factor. The mechanisms involved in the pathophysiology of X-linked hypophosphatemia, Hyp, and oncogenic hypophosphatemic osteomalacia are complex and are the results of mutations in a putative zinc metalloprotease. Inactivation inPHEX gene function initiates a series of events that result in severe perturbations in renal Pi transport and metabolism of vitamin D. There are a number of possible working models that could explain the experimental observations. However, our studies clearly show that a humoral factor (phosphatonin) inhibits the transcription of the type II Na+/Pi cotransporter gene (Fig. 4). Phosphatonin may be a key modulator of phosphate homeostasis.