Article Figures & Data
Figures
- FIG 1
Binding (SPR analysis) of IL-6 to sIL-6R, sSorLA, and the Vps10p-D of SorLA (A to E) and SorLA-mediated uptake of IL-6 in cells (F) and in LAMP-1-positive vesicles (G). The concentration dependence of IL-6 binding to immobilized sIL-6R (A), sSorLA (B), and SorLA Vps10p-D (D) was evaluated. IL-6 was applied at the given concentrations, and the indicated Kd values were calculated on the basis of the collected sum of data. SorLA propeptide-mediated inhibition of IL-6 (100 nM) binding to sSorLA (C) and the SorLA Vps10p-D (E). SorLA was subjected to saturating concentrations of propeptide (2 μM) prior to injection of a mixture of propeptide (2 μM) and IL-6 (100 nM). The projected red lines indicate IL-6 binding obtained in the absence of propeptide, i.e., curves to be expected if the propeptide does not inhibit. (F) Untransfected and SorLA-transfected HEK293 cells were incubated (37°C, 30 min) at 125 nM IL-6 in the absence or presence of 20 μM SorLA propeptide (pro) as indicated. Subsequently, the cells were washed, fixed, permeabilized, and stained using goat anti-IL-6 and mouse anti-SorLA as primary antibodies, as well as Alexa Fluor 488-conjugated donkey anti-goat and Alexa Fluor 568-conjugated donkey anti-rabbit antibodies as secondary antibodies. (G) Immunofluorescence showing accumulation and LAMP-1 colocalization of IL-6 (arrows) in cells treated with lysosomal inhibitors prior to and during 3 h of incubation with IL-6 (Pearson's r = 0.42). Scale bars, 10 μm.
- FIG 2
Uptake of IL-6 in astrocytes is inhibited by SorLA propeptide. (A) Astrocytes isolated from wt and SorLA ko mice were incubated (37°C, 30 min) in unsupplemented medium or in medium supplemented with 125 nM IL-6 with or without 20 μM SorLA propeptide. The cells were then washed prior to fixation and permeabilization before staining with goat anti-IL-6 and mouse anti-SorLA antibodies and the proper secondary antibodies. (B) Histogram showing the average number of IL-6 containing vesicles per cell as determined by automated counting in 15 randomly selected astrocytes. Each column represents mean value, and bars indicate the SEM. The data were evaluated using one-way ANOVA, and post hoc analysis was carried out using Tukey's test. Scale bars, 10 μm.
- FIG 3
SPR analysis of the binding of sIL-6R to sSorLA and to the Vps10p-D of SorLA (A to D) and SorLA-mediated uptake of sIL-6R in cells (E). The concentration dependence of sIL-6R binding to immobilized sSorLA (A) and the SorLA Vps10p-D (C) was evaluated. The indicated Kd values were calculated on the basis of the collected sums of data. The inhibition by SorLA propeptide (pro) of sIL-6R binding to sSorLA (B) and to the Vps10p-D (D) was also evaluated. SorLA was preincubated with a saturating concentration of propeptide (2 μM) prior to the injection of a mixture of propeptide (2 μM) and sIL-6R (100 nM). The response obtained with sIL-6R alone, i.e., the curve to be expected in the absence of inhibition, is indicated in each case in red. (E) Uptake of sIL-6R in SorLA-transfected and wt HEK293 cells. The cells were incubated (37°C, 30 min) at 250 nM sIL-6R in the absence or presence of 20 μM SorLA propeptide. The cells were then washed, fixed, permeabilized, and subsequently stained with mouse anti-IL-6R and rabbit anti-SorLA as primary antibodies and with Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 568-conjugated goat anti-rabbit antibodies as secondary antibodies. Scale bars, 10 μm.
- FIG 4
SorLA accounts for the uptake of sIL-6R in astrocytes. (A) Astrocytes isolated from wt and SorLA ko mice were incubated (37°C, 30 min) in unsupplemented medium or in medium containing 250 nM sIL-6R with or without 20 μM SorLA propeptide (pro) as indicated. The cells were washed, fixed, and permeabilized before staining with mouse anti-IL-6R, rabbit anti-SorLA antibodies, and the matching secondary antibodies. (B) Histogram showing the average number of sIL-6R positive vesicles found in each of nine randomly selected wt and SorLA ko astrocytes. Each column represents the mean value, and bars indicate the SEM. The data were evaluated using one-way ANOVA and Tukey's test. Scale bars, 10 μm.
- FIG 5
Interaction between full-length IL-6R and SorLA in cells. (A) HEK293 cells transfected with SorLA or IL-6R or both receptors in combination were incubated with the non-membrane-permeable chemical cross-linker DTSSP (2 nM). After 45 min, the reaction was stopped, and the receptor proteins were immunoprecipitated from cell lysates with anti-SorLA. Reduced samples of precipitate were analyzed by SDS-PAGE and Western blotting. (B) HEK293 cells transfected with IL-6R or IL-6R/SorLA were biolabeled (4 h, 37°C) using [35S]cysteine and [35S]methionine and then washed and lysed. Subsequently, receptor proteins were immunoprecipitated from cell lysates using anti-IL-6R and analyzed by reducing SDS-PAGE and autoradiography.
- FIG 6
Influence of SorLA and the cytoplasmic IL-6R domain on the internalization and localization of IL-6R in cells. HEK293 transfectants expressing IL-6R (B) or IL-6RΔtail alone (C) or coexpressed with SorLA (A and D) were incubated (4°C, 2 h) with mouse anti-IL6R and rabbit anti-SorLA, washed, and then reincubated at zero time in warm media (37°C) supplemented or not supplemented with 25 nM IL-6. At the indicated times (0 and 25 min), the cells were fixed, permeabilized, and stained with Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 568-conjugated goat anti-rabbit antibodies. (E) HEK293 cells transfected with IL-6R, IL-6R/SorLA, or IL-6R/SorLAΔtail were subjected to subcellular fractionation. The fractions were subjected to Western blotting and probed with antibodies against SorLA, IL-6R, and cd49b (control). Scale bars, 10 μm.
- FIG 7
Cellular half-life and shedding of IL-6R upon coexpression with SorLA. (A, upper panel) HEK293 cells expressing IL-6R or IL-6R/SorLA were biolabeled (4 h, 37°C) with [35S]cysteine and [35S]methionine in the presence of brefeldin A, washed, and incubated in warm medium (37°C). At the indicated time points, IL-6R was immunoprecipitated from cell lysates and analyzed by reducing SDS-PAGE and autoradiography. The lower panel (histograms) shows quantification of band densities (means ± SEM) in three similar experiments. Results are given relative to results obtained at zero time (assigned the value 1). (B) IL-6R and SorLA in medium and cell lysates of HEK293 single and double transfectants. The cells were incubated in culture medium for 0 or 360 min, and the content of IL-6R and SorLA found in the medium (m) and in the cell lysate (l) was detected by Western blotting and quantified by using densitometry of the specific bands. The histogram (B, lower panel) shows the estimated amounts of shed IL-6R (found in the medium) relative to the amount of IL-6R found in the medium of IL-6R single transfectants at 360 min of incubation. Each column represent mean values, and bars indicate the SEM (n = 5).
- FIG 8
IL-6 cis signaling but not trans signaling is affected in SorLA transfectants. (A) BA/F3 cells transfected with gp130 and IL-6R alone or in combination with SorLA were incubated (37°C, 15 min) in the absence or presence of 5 nM IL-6. The levels of total and phosphorylated STAT3 were subsequently determined by Western blotting and densitometry. (B and C) HEK293 cells, with endogenous expression of gp130, and transfected with IL-6R alone or in combination with SorLA (B) or SorLAΔtail (C) were stimulated with IL-6 and probed for STAT3 and pSTAT3 as described above. (D) BA/F3 transfected with gp130 or double transfected with gp130 and SorLA were incubated (37°C, 15 min) in blank medium or medium containing both IL-6 and sIL-6R (5 nM each). The cell lysates were analyzed for STAT3 and pSTAT3 as described above. (E) Wild-type HEK293 expressing gp130 and corresponding SorLA transfectants were stimulated and analyzed as in panel D. The left panels (A to E) show Western blot results of single experiments; the right panels sum up the results of several experiments and show results obtained in SorLA transfectants (open columns) relative to values obtained in cells not transfected with SorLA (shaded columns). Each column represents the mean values, and bars indicate the SEM (A, n = 9; B, n = 6; C, n = 6; D, n = 4; E, n = 7). P values were calculated using a Wilcoxon signed-rank test based on raw data. (F) SPR analysis of the binding of IL-6 to immobilized sIL-6R in the presence of a surplus of sSorLA. Soluble IL-6R was subjected to sSorLA (1 μM) prior to the injection of a mixture of sSorLA (1 μM) and IL-6 (100 nM). The response obtained with IL-6 (100 nM) alone is shown.
- FIG 9
Soluble SorLA may stabilize IL-6 trans signaling. (A) BA/F3 cells expressing gp130 were stimulated (15 min, 37°C) as indicated, and the resulting levels of pSTAT3/STAT3 were determined by Western blotting of cell lysates. Prior to stimulation, sIL-6R (5 nM) and sSorLA (40 nM) were incubated separately (3 h, room temperature), whereas IL-6 (5 M) was preincubated alone or in combination with 40 nM sSorLA (sSorLA:IL-6). The left panel shows a Western blot of a representative experiment. The right panel summarizes results of several (n) experiments in which the pSTAT3 levels (measured by densitometry) were set relative to the level obtained in response to IL-6+IL-6R (assigned the value 1). Bars indicate the SEM, The P value was calculated using the Wilcoxon signed-rank test. (B) The same BA/F3 cells were stimulated (15 min, 37°C) as indicated, but in this case none of the reagents had been coincubated prior to stimulation. The relative pSTAT3 levels were determined as described above. The inset depicts a Western blot of a single experiment, and the histogram summarizes results of nine experiments. (C) pSTAT3 levels in the same cells and stimulated as for panel A except that sSorLA had been substituted with sSorCS3. The inset shows a Western blot of a single experiment, the histogram summarizes (as described above) results of five separate experiments. (D) pSTAT3 levels in SorLA ko astrocytes stimulated with preincubated reagents as for panel A. The columns represent mean values (± SEM, n = 3) relative to the pSTAT3 level in unstimulated astrocytes (assigned value 1). Data were evaluated by using one-way ANOVA and Tukey's test. (E) SPR analysis of the binding of sIL-6R to a preformed sSorLA:IL-6 complex. Immobilized sSorLA was initially exposed to IL-6 (100 nM) prior to the injection of fresh buffer containing 100 nM sIL-6R. The subsequent increase in response units signifies the binding of sIL-6R to the preformed sSorLA:IL-6 complex. The estimated Kd is indicated.
