Figure 1.
Constitutive shedding of human Ob-R isoforms.
(A) sOb-R concentration in the supernatant of Ob-R transfected HEK cells after incubation for 48 h under normal culture conditions (37°C, 5% CO2). VC, vector control (pcDNA 3.1) (B) Ob-R in whole cell lysates of Ob-R transfected HEK cells was determined by an in-house immunofunctional assay. Ob-R protein expression is presented realtive to Ob-R levels of Ob-Rfl transfected cells. (C) I125-leptin binding of Ob-R transfected HEK cells. Ob-R transfected cells were incubated for 48 h under normal culture conditions (37°C, 5% CO2) and I125-leptin binding assay was performed. I125-leptin binding is shown in % relative to amount of used I125-leptin tracer. (D) Normalisation of sOb-R (A) to I125-leptin binding (C). Data are presented as means ± SD of n≥3 experiments; n.d., not detectable.
Figure 2.
Constitutive shedding of human Ob-R isoforms is mainly mediated by ADAM10.
(A) sOb-R levels in the supernatant of Ob-R219.3 transfected HEK cells after incubation with the metalloprotease inhibitors GI/GW (3 µM) or DMSO (0.1%) for 24 h. sOb-R levels of GI/GW incubated cells are presented relative to those of cells incubated with DMSO. (B and C) ADAM10 and ADAM17 mRNA (B) and protein expression (C) in lysates of HEK cells transfected with a specific siRNA for ADAM10 or ADAM17 or a non-targeting siRNA (50 nM). (D and E) sOb-R levels in the supernatant of HEK cells transfected with a specific siRNA for ADAM10, ADAM17 or a non-targeting siRNA (50 nM) and subsequently with Ob-Rfl (D) or Ob-R219.3 (E) followed by a further incubation under normal culture conditions for 24–72 h. sOb-R levels of ADAM10/ADAM17 siRNA transfected cells are shown relative to those of cells transfected with a non-targeting siRNA. Data are presented as means ± SD of n≥3 experiments.
Figure 3.
PKC activation leads to an increased release of sOb-R by Ob-R transfected cells, mainly mediated by ADAM10.
(A) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with PMA (10 ng/ml) or DMSO (0.1%) for 24 h. sOb-R levels of PMA-treated cells are displayed relative to sOb-R levels of DMSO-treated cells. (B) sOb-R levels in the supernatant of Ob-R219.3 transfected cells after incubation with PMA (10 ng/ml) and or staurosporine (0.01 µM) or DMSO (0.1%) for 24 h. sOb-R levels of PMA- and staurosporine-treated cells are shown relative to those of cells treated with DMSO. (C, D) sOb-R levels in the supernatant of HEK cells transfected with a specific siRNA for ADAM10 and or ADAM 17 or a non-targeting siRNA (50 nM) and subsequently with Ob-Rfl (C) or Ob-R219.3 (D) followed by a further incubation for 24–72 h with PMA (10 ng/ml). sOb-R levels of ADAM10/ADAM17 siRNA transfected cells are presented relative to sOb-R levels of cells transfected with a non-targeting siRNA. Data are presented as means ± SD of n≥3 experiments.
Figure 4.
Caspase-mediated apoptosis, induced by incubation with staurosporine, activates Ob-R shedding mainly mediated by ADAM10.
(A) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with staurosporine (0.1–0.5 µM) or DMSO (0.1%) for 24 h and 48 h. sOb-R levels of staurosporine-treated cells are displayed relative to sOb-R levels of cells treated with DMSO (B) sOb-R levels in the supernatant of Ob-R219.3 transfected cells after pre-incubation with the broad spectrum caspase inhibitor Z-VAD(OMe)FMK (100 µM) or DMSO (0.1%) for 30 min and subsequent stimulation with staurosporine (0.1–0.5 µM) or DMSO (0.1%) for 24 h and 48 h. sOb-R levels of staurosporine- or Z-VAD(OMe)FMK-treated cells are shown relative to those of cells treated with DMSO. (C) Representative western blot of cleaved caspase-3 and cleaved PARP after staurosporine incubation. Ob-R219.3 transfected cells were pre-incubated with Z-VAD(OMe)FMK (100 µM) or DMSO (0.1%) for 30 min and subsequently stimulated with staurosporine (0.1–0.5 µM) or DMSO (0.1%) for 48 h. Following this incubation cleaved caspase-3 and cleaved PARP in cell lysates was determined by western blot analysis. PC, positive control (cell lysate of Jurkat cells treated with cytochrome c). (D) sOb-R levels in the supernatant of HEK cells transfected with a non-targeting, ADAM10 or ADAM17 specific siRNA (50 nM) and subsequently with Ob-R219.3 after incubation with staurosporine (0.5 µM) or DMSO (0.1%) for 48 h. sOb-R levels of ADAM10 and ADAM17 siRNA transfected cells are presented relative to sOb-R levels of cells transfected with a non-targeting siRNA and treated with DMSO. Data are presented as means ± SD of n≥3 experiments.
Figure 5.
Enhanced release of sOb-R after palmitate incubation is mediated by ADAM10 and ADAM17 and inhibited by oleate.
(A) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with palmitate (0.5–1 mM), oleate (0.5 mM–1 mM or methanol (2%) for 24 and or 48 h. sOb-R levels of palmitate- or oleate-treated cells are displayed relative to those of cells treated with methanol. (B) sOb-R levels in the supernatant of HEK cells transfected with a non-targeting, ADAM10 or ADAM17 specific siRNA (50 nM) and subsequently with Ob-R219.3, after incubation with palmitate (1 mM) or methanol (2%) for 48 h. sOb-R levels of ADAM10 and ADAM17 siRNA transfected cells are shown relative to sOb-R levels of cells transfected with a non-targeting siRNA and treated with methanol. (C) Representative western blot of cleaved PARP after palmitate incubation. Ob-R transfected cells were incubated with palmitate (0.5–1 mM) or Methanol (2%) for 24 and 48 h. Following this incubation cleaved PARP in cell lysates was determined by western blot analysis. (D) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with palmitate 1 mM and Z-VAD 100 µM or methanol (2%) for 48 h. sOb-R levels of palmitate- and or Z-VAD-stimulated cells are displayed relative to those of cells treated with methanol. (E) Representative western blot of cleaved caspase-3 and cleaved PARP. Ob-R transfected cells were incubated with palmitate (0.5–1 mM), palmitate (0.5–1 mM) and Z-VAD (100 µM) or Methanol (2%) for 48 h. Following this incubation cleaved caspase-3 and cleaved PARP in cell lysates was determined by western blot analysis. (F and G) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with palmitate (1 mM), oleate (0.1–0.5 mM), palmitate (1 mM) and oleate (0.1–0.5 mM) or methanol (2%) for 48 h. sOb-R levels of palmitate-, oleate– or palmitate- and oleate-treated cells are displayed relative to those of cells treated with methanol. (H) Representative western blot of cleaved PARP after co-incubation with palmitate and oleate. Ob-R transfected cells were incubated with palmitate (0.5–1 mM), palmitate (0.5–1 mM) and oleate (0.1–0.5 mM) or Methanol (2 %) for 48 h. Following this incubation cleaved PARP in cell lysates was determined by western Blot analysis. Data are presented as means ± SD of n≥3 experiments (Fig. 5 D and E, n = 2).
Figure 6.
Co-expression of Ob-R219.3 to Ob-Rfl and increased sOb-R levels modulate leptin signaling of Ob-Rfl.
(A) Ob-R transfected cells were serum-starved for 16 h and subsequently incubated with leptin (100 ng/ml) for 30 min. P-STAT3 and total STAT3 levels in lysates of HEK cells transfected with equimolar amounts of Ob-Rfl + GFP (equimolar to Ob-R219.3) or Ob-Rfl + Ob-R219.3 were determined by ELISA and western blot. P-STAT3/total STAT3 ratios are displayed relative to P-STAT3/total STAT3 ratio of non-treated cells transfected with Ob-Rfl + GFP. (B) P-STAT3 and total STAT3 levels in lysates of HEK cells transfected with Ob-Rfl. Ob-Rfl-transfected cells were stimulated with media containing leptin (10 ng/ml) and increasing concentrations of sOb-R (0–1000 ng/ml). P-STAT3 and total STAT3 levels are displayed relative to those of leptin-treated control without sOb-R. Data are presented as means ± SD of n≥2 experiments.
Figure 7.
Leptin inhibits sOb-R release from Ob-Rfl or Ob-R219.3 transfected cells into the supernatant.
Ob-Rfl or Ob-R219.3 transfected cells were incubated with leptin (5–100 ng/ml) for 24 h. Following this incubation sOb-R in the supernatant was determined. sOb-R levels of leptin-treated cells are shown relative to those of non-treated cells. Data are presented as means ± SD of n≥3 experiments.
Figure 8.
ER stress causes downregulation of membrane Ob-R, impaired leptin signaling via Ob-Rfl and decreased sOb-R concentrations.
(A) Representative western blot for protein expression of ER stress markers BiP (GRP78), CHOP (GADD153) and PDI after treatment of Ob-R transfected HEK cells with tunicamycin (3 µg/ml) or DMSO (0.1%) for 4–24 h. (B) Ob-Rfl transfected cells were incubated with tunicamycin (3 µg/ml) or DMSO (0.1%) for 4–24 h and I125-leptin binding assay was performed. I125-leptin binding of tunicamycin-treated samples is displayed relative to that of non-treated controls. (C and D) P-STAT3 (C) and total STAT3 (D) levels in lysates of Ob-Rfl transfected cells after incubation with tunicamycin (3 µg/ml) or DMSO (0.1%) for 4–24 h and subsequent stimulation with leptin (100 ng/ml) for 30 min. P-STAT3 and total STAT3 levels of leptin-stimulated cells are displayed relative to that of non-treated controls. (E) sOb-R levels of Ob-Rfl or Ob-R219.3 transfected cells after incubation with tunicamycin (3–20 µg/ml) or DMSO (0.2%) for 24 and 48 h. sOb-R levels of tunicamycin-treated cells are displayed relative to those of non-treated cells. Data are presented as means ± SD of n≥3 experiments (Fig. 8 A, n = 1; Fig. 8 B, n = 2).