Glycosylation of Sodium/Iodide Symporter (NIS) Regulates Its Membrane Translocation and Radioiodine Uptake

<div><p>Purpose</p><p>Human sodium/iodide symporter (hNIS) protein is a membrane glycoprotein that transports iodide ions into thyroid cells. The function of this membrane protein is closely regulated by post-translational glycosylation. In this study, we measured glycosylation-mediated changes in subcellular location of hNIS and its function of iodine uptake.</p><p>Methods</p><p>HeLa cells were stably transfected with hNIS/tdTomato fusion gene in order to monitor the expression of hNIS. Cellular localization of hNIS was visualized by confocal microscopy of the red fluorescence of tdTomato. The expression of hNIS was evaluated by RT-PCR and immunoblot analysis. Functional activity of hNIS was estimated by radioiodine uptake. Cyclic AMP (cAMP) and tunicamycin were used to stimulate and inhibit glycosylation, respectively. In vivo images were obtained using a Maestro fluorescence imaging system.</p><p>Results</p><p>cAMP-mediated Glycosylation of NIS resulted in increased expression of hNIS, stimulating membrane translocation, and enhanced radioiodine uptake. In contrast, inhibition of glycosylation by treatment with tunicamycin dramatically reduced membrane translocation of intracellular hNIS, resulting in reduced radioiodine uptake. In addition, our hNIS/tdTomato fusion reporter successfully visualized cAMP-induced hNIS expression in xenografted tumors from mouse model.</p><p>Conclusions</p><p>These findings clearly reveal that the membrane localization of hNIS and its function of iodine uptake are glycosylation-dependent, as our results highlight enhancement of NIS expression and glycosylation with subsequent membrane localization after cAMP treatment. Therefore, enhancing functional NIS by the increasing level of glycosylation may be suggested as a promising therapeutic strategy for cancer patients who show refractory response to conventional radioiodine treatment.</p></div

Changes of radioiodine uptake by regulation of glycosylation.

<p>(A) Glycosylation inhibitor, tunicamycin reduced radioiodine (¹²⁵I) uptake in the hNIS/tdTomato expressing cells. (B) Glycosylation activator, cAMP increased radioiodine (¹²⁵I) uptake in the hNIS/tdTomato expressing cells. Accumulation of radioiodine was measured with a gamma-counter at 24 hr and 72 hr after treatment (*, P<0.05; **, P<0.01; ***, P<0.001; N = 3).</p

Generation of HeLa cells expressing the hNIS/tdTomato fusion gene.

<p>(A) Schematic representation of the hNIS/tdTomato fusion gene reporter construct (upper). RT-PCR (lower left) and Immunoblot analysis (lower right) showed stable expression of hNIS/tdTomato gene in HeLa cells. (B) Fluorescence microscope image shows the hNIS/tdTomato fusion protein expression in HeLa-hNIS/tdTomato cells. Cellular hNIS proteins were imaged using a time-lapse live cell imaging system (upper) and confocal microscopy (lower). (C) Red fluorescence from HeLa-hNIS/tdTomato cells increased with increasing cell number. (D) Function of hNIS/tdTomato in HeLa cells was measured by I-uptake and iodine uptake shows cell number dependency.</p

Enhancement of red fluorescence from cAMP treated HeLa-hNIS/tdTomato cells in mouse.

<p>(A) HeLa-hNIS/tdTomato cells (1x10⁵) were treated with cAMP (0–100 μM) at the indicated concentrations. Red fluorescent signals of cell pellets were imaged and measured using Maestro fluorescence imaging system. (B) The red fluorescent intensity that represents the expression of hNIS/tdTomato protein increased in proportion to the concentration of cAMP. (C) HeLa-hNIS/tdTomato cells were cultured with cAMP for 72 hr and then transplanted into the right flanks of mice. Untreated HeLa-hNIS/tdTomato cells were transplanted into the left flanks of mice. Red fluorescence of HeLa-hNIS/tdTomato was imaged by Maestro^TM. (D) Red fluorescent intensity of HeLa-hNIS/tdTomato cells was analyzed by the Maestro^TM software program. Bars represent mean ± SD (*, P<0.05; N = 3).</p

Membrane translocation of hNIS protein by cAMP after inhibition of de novo protein synthesis.

<p>To inhibit cAMP-induced protein synthesis, AMD (5 ng/mL) or CHX (1 μg/ml) were pretreated 24h before treatment of 100 μM cAMP. (A) Enhanced expression of hNIS/tdTomato proteins by cAMP was visualized after blocking de novo protein synthesis. (B) Enhanced membrane localization of hNIS/tdTomato proteins by cAMP was visualized after blocking de novo protein synthesis. Red fluorescent intensity was analyzed with MetaMorph software. An arbitrary threshold that represented the cytosolic compartment was designated. Threshold intensity of fluorescence was adjusted to show membrane-localized hNIS/tdTomato protein only. Signals over or under the threshold were depicted as red or gray, respectively. (C) The upper threshold of red fluorescent intensity was measured to quantify the membrane localized hNIS/tdTomato protein. Confocal images were collected from at least three different regions of each sample. Bars represent mean ± SD (*, P<0.05; **, P<0.01; N = 3).</p

Membrane localization of glycosylated hNIS/tdTomato protein.

<p>(A) HeLa-hNIS/tdTomato cells were treated with tunicamycin (1.2 μM) or cAMP (100 μM), and the red fluorescent signals were photographed using confocal microscopy. Based on a cross-sectional analysis using fluorescence profiling of MetaMorph software, an arbitrary threshold that represented the cytosolic compartment was designated. Signals over the threshold were considered to be from the membrane compartment. (B) NIS expression was observed by immunoblot analysis with cellular protein extracts (20 μg) from tunicamycin- and cAMP-treated HeLa-hNIS/tdTomato cells. β-actin was used as an internal control. (C) NIS expression was observed by immunoblot analysis with membrane proteins isolated from cAMP-treated cells. Caveolin was used as an internal control.</p

Visualization and image-based quantification of membrane-localized hNIS/tdTomato protein.

<p>(A) HeLa-hNIS/tdTomato cells were treated with tunicamycin or cAMP at the indicated concentrations, and red fluorescent signals were photographed using confocal microscopy. Based on a cross-sectional analysis using fluorescence profiling of MetaMorph software, an arbitrary threshold that represented the cytosolic compartment was designated. Signals over or under the threshold were depicted as red or gray, respectively. At least three different regions of each sample were imaged. Confocal images were collected from at least three different regions of each sample. (B) Relative fluorescence signal intensities of hNIS/tdTomato proteins after the treatment of glycosylation inhibitor (tunicamycin). (C) Relative fluorescence signal intensities of hNIS/tdTomato proteins after the treatment of glycosylation activator (cAMP). Fluorescent signal intensities acquired from threshold images were measured for quantification of membrane-localized hNIS/tdTomato proteins after glycosylation inhibitor or activator treatment. Relative fluorescence signal intensities were calculated based on the fluorescence intensity of non-treated control. Bars represent mean ± SD (*, P<0.05; **, P<0.01; N = 3).</p

<i>In Vivo</i> Bioluminescence Imaging for Prolonged Survival of Transplanted Human Neural Stem Cells Using 3D Biocompatible Scaffold in Corticectomized Rat Model

<div><p>Stem cell-based treatment of traumatic brain injury has been limited in its capacity to bring about complete functional recovery, because of the poor survival rate of the implanted stem cells. It is known that biocompatible biomaterials play a critical role in enhancing survival and proliferation of transplanted stem cells via provision of mechanical support. In this study, we noninvasively monitored <i>in vivo</i> behavior of implanted neural stem cells embedded within poly-l-lactic acid (PLLA) scaffold, and showed that they survived over prolonged periods in corticectomized rat model. Corticectomized rat models were established by motor-cortex ablation of the rat. F3 cells expressing enhanced firefly luciferase (F3-effLuc) were established through retroviral infection. The F3-effLuc within PLLA was monitored using IVIS-100 imaging system 7 days after corticectomized surgery. F3-effLuc within PLLA robustly adhered, and gradually increased luciferase signals of F3-effLuc within PLLA were detected in a day dependent manner. The implantation of F3-effLuc cells/PLLA complex into corticectomized rats showed longer-lasting luciferase activity than F3-effLuc cells alone. The bioluminescence signals from the PLLA-encapsulated cells were maintained for 14 days, compared with 8 days for the non-encapsulated cells. Immunostaining results revealed expression of the early neuronal marker, Tuj-1, in PLLA-F3-effLuc cells in the motor-cortex-ablated area. We observed noninvasively that the mechanical support by PLLA scaffold increased the survival of implanted neural stem cells in the corticectomized rat. The image-guided approach easily proved that scaffolds could provide supportive effect to implanted cells, increasing their viability in terms of enhancing therapeutic efficacy of stem-cell therapy.</p></div

<i>In vivo</i> bioluminescence imaging of the implanted F3-effLuc/PLLA scaffold in a corticectomized rat model.

<p>(A) After F3-effLuc cells were incubated within the PLLA scaffold for 2 hr, the cell/scaffold complex was implanted into the ablated motor cortex area of the rat brain. Firefly luciferase bioluminescence imaging was performed over 14 days. The prolonged luminescence signals in F3-effLuc cells within the PLLA scaffold were clearly visualized in the ablated area. (B) Quantitative ROI analysis showed significantly enhanced survival duration for F3-effLuc cells within the PLLA scaffold (n = 6). P value, * <0.005.</p

Validation of stem cell characteristics in F3 cells and F3-effLuc cells.

<p>Flow cytometric analysis showed F3 and F3-effLuc cells are positive for the stem cell surface marker, (a) CD44, and the intracellular marker s, (b) Nestin, (c) Ki67, (d) Sox1, and (e) Sox2.</p