Fig 1.
Uptake and transepithelial transport of 64Cu from human ceruloplasmin (Cp) by monolayers of a human mammary epithelial cells (PMC42).
PMC42 monolayers with tight junctions (resistance > 300 Ohms), grown in bicameral chambers, were exposed to 64Cu-Cp purified from secretions of HepG2 cells treated with 64Cu-NTA, on the basolateral (“blood”) side, and radioactivity (cpm) in washed cells (“Cells”) and apical fluid (“AF”) was measured. Total Cu uptake (“Total”) was the sum of radioactivity in cells plus AF. (A) Total uptake, and radioactivity in cells and AF after 1 and 16 h of incubation. (B) Total uptake and cell accumulation of 64Cu from Cp after 1 h, in the absence and presence of Ag(I) (50 μM). Data are Means ± SD, N = 4. *p<0.01 for Ag effect.
Fig 2.
Uptake of 64Cu from mouse Cp by mouse embryonic fibroblasts (MEF) expressing (Ctr1+/+; WT) and not expressing (Ctr1-/-; Null) Ctr1, and effects of Ag(I) or cooling to 2° C.
(A) Time course of uptake over 6 h, measured in terms of cpm per μg cell protein, showing results for cells in individual 6 well plates (representative data for multiple studies). The difference in slope of the lines for the WT and null cells was statistically significant (p<0.001) by model-validated regression analysis (see Methods). (B) Effect of Ag(I) (5 μM) on the uptake rate of Cu from Cp over 3 h (%dose/h/mg cell protein). Means ± SD (N = 3). [A lower concentration of Ag(I) was used than for the PMC42 cells (Fig 1B) because the MEFs lifted off the plate at higher concentration.] (C) Effect of cooling cells to 2° C during measurements of uptake over 1 h, Means ± SD (N = 6–9). *p <0.001 for difference from 37° C for both cell types.
Fig 3.
Effects of extensive washing at different pHs, and of trypsin, on retention of 64Cu from mouse Cp by mouse embryonic fibroblasts expressing (Ctr1+/+, WT; dark bars) and not expressing (Ctr1-/-, Null; light bars) Ctr1.
(A) Combined data from several studies, showing radioactivity in washes and cells 3 h after 64Cu-Cp exposure, after washing extensively with phosphate buffered saline or MEM (without FBS), pH 7.0. The first wash contained 92% of the initial dose. Additional washes (beyond 4) made no difference. (B and C) Effects of washing cells 3X with MEM and then with buffers of lower pH (vs MEM): acetate buffered saline, pH 5.0 (B) or pH 3.0 (C). (D) Effects of incubating with trypsin (3%, concentrations– 3X higher than for cell culturing) over 5 or 60 min, on retention of 64Cu radioactivity by fibroblasts previously exposed to 64Cu-Cp for 3 h. Data are Means ± SD for N = 3–6 (A), N = 3 (B-D).
Fig 4.
Internalization of Cp copper upon incubation with WT and Ctr1 null fibroblasts with Cp.
(A) Cells were exposed to purified mouse 64Cu-labeled Cp for 3 h, and radioactivity (% dose) in the cell cytosol and pellet of washed cells was measured after disruption and centrifugation for 1 h at 105,000 x g. Combined data (Means ± SD; N = 8) from four experiments for both kinds of cells; cell disruption for three experiments was by nitrogen bomb cavitation, one was by exposure to hypotonic buffer. (B) Actual (non-radioactive) copper accumulated by human mammary epithelial cells (PMC42) or mouse embryonic fibroblasts (MEF; Ctr1+/+), after 6 or 24 h incubation with purified human or mouse Cp, respectively. Prior to Cp exposure, cells were pretreated for 48 h with Teta (120 μM) or BCS (1 mM), respectively, to induce copper deficiency. Controls (Contr) were cells treated only with chelator; Cp were cells treated with Cp-Cu after pretreatment with chelator; Untr refers to cells not treated with chelator but kept in normal MEM medium (without FBS); Ovalb refers to chelated cells treated with 1 mg/ml ovalbumin as another kind of control. Data are cellular Cu concentrations per mg cell protein, calculated as percent of controls in the same experiment, presented as Means ± SD (N = 3–5) for the PMC42 cells, and Means ± AD for the MEF (results of two separate individual experiments). *p <0.001 for difference from controls.
Fig 5.
Rates of Cu uptake from Cp as a function of Cp-Cu concentrations.
Purified mouse 64Cu-Cp samples, used to measure rates of 64Cu uptake from Cp by mouse embryonic fibroblasts (expressing and not expressing Ctr1), were assayed for Cu (Cp Copper; μM) and/or for pPD oxidase activity (Cp Oxidase Activity; nmol/min/mg cell protein) to provide measures of the amounts of holo Cp present. Uptake rates (%dose/h/mg cell protein) on the y-axis were plotted against Cp Copper concentrations (A) or Cp Oxidase Activity (B) on the x-axes. Independent data for Ctr1+/+ and Ctr1-/- cells are shown, points and error bars indicating Means ± SD (N = 3). Normal physiological (plasma) concentrations of Cp copper and oxidase activity in mice are indicated in the upper left corner of the graphs to compare with amounts used in the uptake assays. [Note that Cp-Cu concentrations are 2-3-fold higher in humans than mice.] For the WT (open squares) in both A and B, concentrations between the central values differed significantly from those that were higher or lower by two-tailed t-test (p<0.05–0.01). For the Ctr1-null cells, rates of uptake at the highest Cp concentrations were significantly greater than those at the lower concentrations (p<0.01). By regression analysis (with model validation), the slopes of the lines in both (A) and (B) were significantly positive (p <0.01) i.e. increased Cp correlated with increased rates of uptake.
Fig 6.
No inhibition of Cu uptake from Cp by inhibitors of endocytosis at standard concentrations: nocodazole (Noc or N; 10 μM), FSBA (F; 100 μM), Pitstop (Pit; 12 μM), or DynaSore© (80 μM).
Cells were preincubated with the inhibitors for 1 h before adding the 64Cu-Cp. Values are uptake rates as percent of those for controls (Con) in the same experiment, Means ± SD (N = 9–7 for Con and Noc; 3–5 for the other treatments. Actual uptake rates of control (untreated cells) ranged from 1 to 2.5 percent dose/h/mg cell protein, dose being the 64Cu in Cp. Only Noc alone had a statistically significant effect compared to the control, and it was stimulatory.
Fig 7.
Conversion of holo Cp to apo Cp during uptake of Cu from Cp by MEF cells.
(A) and (B) Purified non-radioactive mouse Cp was incubated in MEM for 24 h, without or with MEF cells expressing Ctr1, following which varying volumes of the Cp in the medium were analyzed for the proportion of apo to holo Cp by native PAGE Western blotting. High range prestained BioRad electrophoresis standards (Std) were included to identify the HoloCp and ApoCp bands. The “115” and “78” kDa standards are visible on the blots. (ApoCp migrates considerably slower than the 115 k Std; HoloCp migrates at or above the 78 k Std.) (A) Examples of Westerns. (B) Densitometric data (Means ± SD, N = 5 and 8) for blots from 4 studies, showing decrease in holo Cp and increase in apo Cp when incubated with versus without cells, *p <0.01 for difference. (C) Example of stained SDS-PAGE gel (above) and Western (below), and (D) summary of these and other densitometric data (SDS-PAGE Westerns that measure total Cp protein—both apo and holo) to determine whether exposure to cells changed/lowered total Cp concentrations over 24 h. Data are Means ± SD, N = 6. There was no statistically significant difference.
Fig 8.
Inhibition of uptake of 64Cu from Cp by an excess of non-radioactive ionic Cu(II), Cu(I), or Fe(III).
Uptake of 64Cu from purified mouse Cp over 3 h by MEF cells expressing and not expressing Ctr1 was measured in the absence and presence of non-radioactive 150 μM Cu(II) and Cu(I) (A) or Fe(III) (B) as the 5:1 NTA:metal complex. Results for cells with and without Ctr1 were similar and so were pooled, and are given as Means ± SD, N = 12 for Cu(II), 6 for Cu(I), and 6 for Fe(III). *p <0.001 for difference from controls.
Fig 9.
Expression of reductase mRNAs by MEF cells, and effect of Steap 2 siRNA knockdown on mRNA and copper reductase activity.
(A) WT and Ctr1-null MEF cells were analyzed for expression of mRNA for dCytb, and Steaps 2, 3 and 4 by quantitative PCR, relative to 18S rRNA [expression = 2^(-∆Ct)]. (B) Effect of Steap 2 siRNA treatment (2 x over 5–6 days) on Steap 2 mRNA levels (relative to 18S RNA) compared to that of scrambled siRNA (ScrRNA), and on cell surface Cu(II) reductase activity, pooling results from two separate experiments, thus given as percent (N = 3 for each) of control (untreated) cells in the same experiment. Data are Means ± SD, N = 6. *p<0.001 for difference from control (ScrRNA).