by Martha B. Johnson, and Caroline A. Enns Diferric transferrin regulates transferrin receptor 2 protein stability by Martha B. Johnson, and Caroline A. Enns Blood Volume 104(13):4287-4293 December 15, 2004 ©2004 by American Society of Hematology

TfR2 increases after the addition of diferric Tf to the medium of HepG2 cells. TfR2 increases after the addition of diferric Tf to the medium of HepG2 cells. (A) TfR2 increases in a time-dependent manner. HepG2 cells were cultured for 4 to 72 hours after the addition of 25 μM diferric Tf or HBS to the medium. Lysates (20 μg total protein) were transferred to nitrocellulose, probed for TfR2 and Tf, and visualized by chemiluminescence. The increase in TfR2 was paralleled by an increase in Tf associated with the cells. (B) TfR2 returns to basal levels after withdrawal of Tf from the medium. HepG2 cells were cultured for 24 hours in the presence of 25 μM diferric Tf, then chased in medium for 0 to 8 hours. TfR2 and Tf levels were analyzed by Western blot using HRP-conjugated secondary antibodies and chemiluminescence. (C) The response of TfR2 to diferric Tf is concentration dependent. HepG2 cells were cultured for 24 hours after the addition of 0 to 25 μM diferric Tf to the medium, and lysates (20 μg total protein) were analyzed by Western blot with fluorescence-labeled secondary antibodies for quantification and HRP-conjugated secondary antibodies for chemiluminescence imaging, as described in “Materials and methods.” The intensity of each band was normalized to the intensity of the 0 μM Tf sample. The log of the normalized intensity is plotted as a function of diferric Tf concentration. The increase in TfR2 is half-maximal at approximately 2.5 μM diferric Tf. (D) TfR2 does not increase in response to nontransferrin-bound iron. TfR2, TfR1, and Ft protein levels were assessed by Western blots of lysates (20 μg total protein) from HepG2 cells cultured for 24 hours in the presence of 100 μM FeNTA (lane 1,+) or 4 mM NTA (lane 2,-). Bands were detected by chemiluminescence. Ft heavy and light chains are visible as a doublet in the lower panel. (E) TfR2 does not increase in response to apo Tf. HepG2 cells were incubated in medium containing 25 μM diferric Tf (lane 1, labeled holo Tf) or apo Tf (lane 3) for 24 hours. Lysates (20 μg total protein) were analyzed by Western blot for TfR2, TfR1, and Ft protein. Bands were detected by chemiluminescence. Martha B. Johnson, and Caroline A. Enns Blood 2004;104:4287-4293 ©2004 by American Society of Hematology

TfR2 increases in HuH7 cells, but not in K562 or TRVb2 cells. TfR2 increases in HuH7 cells, but not in K562 or TRVb2 cells. Cells were cultured for 24 hours after the addition of 25 μM diferric Tf to the medium. The level of TfR2 in lysates from HuH7 (50 μg), K562 (20 μg), and TRVb2 (10 μg) cells was determined by Western blot with chemiluminescence detection. In cells endogenously expressing TfR2, treatment with diferric Tf increased TfR2 in HuH7 human hepatoma cells but not in K562 erythroleukemia cells. TRVb cells stably transfected with TfR2 (TRVb2) did not respond to diferric Tf. Martha B. Johnson, and Caroline A. Enns Blood 2004;104:4287-4293 ©2004 by American Society of Hematology

Regulation of TfR2 occurs at the protein level. Regulation of TfR2 occurs at the protein level. (A) TfR2 transcript in HepG2 cells does not increase in response to diferric Tf. Total RNA was isolated from approximately 1 × 107 HepG2 cells 24 hours after the addition of 25 μM diferric Tf or an equal volume of HBS to the medium. Expression of TfR2, TfR1, and GAPDH transcripts was measured by real-time qRT-PCR analysis of cDNA synthesized from 2 μg total RNA. Levels of TfR2 and TfR1 transcripts are shown relative to GAPDH levels. The graph represents the mean of 3 separate experiments in which each sample was analyzed twice in triplicate. Error bars depict SD. (B) Diferric Tf stabilizes TfR2 protein. HepG2 cells seeded at 2 × 104 cells/cm2 were incubated in normal medium or medium with 25 μM diferric Tf for 24 hours before the addition of 100 μg/mL cycloheximide for 4, 3, 2, 1, 0.5, and 0 hours. Cells were solubilized, lysates from duplicate wells were pooled, and half of each sample was analyzed by Western blot. TfR2 and TfR1 were detected with fluorescence-labeled secondary antibodies for quantification and then with HRP-conjugated secondary antibodies for chemiluminescence imaging. The integrated intensity of TfR2 was normalized to that of TfR1, which did not change detectably over the time-course of the experiment. The normalized intensity was expressed as a percentage of the normalized intensity at time 0, and the log of this value was plotted. Half-life was determined by linear regression analysis. The graph shows the mean of 3 experiments. Error bars indicate average deviation from the mean. Martha B. Johnson, and Caroline A. Enns Blood 2004;104:4287-4293 ©2004 by American Society of Hematology

Down-regulation of TfR1 reduces the increase in TfR2 protein. Down-regulation of TfR1 reduces the increase in TfR2 protein. (A) The 42/6 anti-TfR1 antibody interacts with TfR1 but not with TfR2. HepG2 cell lysates were immunoprecipitated with (+) or without (-) 42/6 anti-TfR1, 3B82A1 anti-TfR1, or 9F81C11 anti-TfR2 monoclonal antibodies and were analyzed by Western blot with sheep anti-TfR1/Tf serum and rabbit anti-TfR2 serum. Bands corresponding to TfR2, TfR1, Tf, and the immunoglobulin heavy chains (Ig HC) are indicated. (B-C) Treatment with 42/6 diminishes the effect of diferric Tf on TfR2. Anti-TfR1 antibody 42/6 was added to the medium of HepG2 cells at a concentration of 25 μg/mL 4 hours before the addition of 25 μM diferric Tf to the medium for 24 hours. To control for possible effects of iron deprivation, a second set of cells was treated identically, but 100 μM FeNTA was added concomitantly with 42/6 antibody. Lysates (20 μg total protein) were analyzed by Western blot. (B) TfR1 and TfR2 bands were visualized with fluorescence-labeled secondary antibodies. (C) Fluorescence imaging was used to quantitate TfR1 and TfR2 protein levels. The integrated intensity of each band is expressed as a percentage of control. The graph depicts the mean ± SD of 4 independent experiments. *P < .05, as determined by Student one-tailed paired t test. Martha B. Johnson, and Caroline A. Enns Blood 2004;104:4287-4293 ©2004 by American Society of Hematology