TLR ligand-induced podosome disassembly in dendritic cells is ADAM17 dependent

Toll-like receptor (TLR) signaling induces a rapid reorganization of the actin cytoskeleton in cultured mouse dendritic cells (DC), leading to enhanced antigen endocytosis and a concomitant loss of filamentous actin–rich podosomes. We show that as podosomes are lost, TLR signaling induces prominent focal contacts and a transient reduction in DC migratory capacity in vitro. We further show that podosomes in mouse DC are foci of pronounced gelatinase activity, dependent on the enzyme membrane type I matrix metalloprotease (MT1-MMP), and that DC transiently lose the ability to degrade the extracellular matrix after TLR signaling. Surprisingly, MMP inhibitors block TLR signaling–induced podosome disassembly, although stimulated endocytosis is unaffected, which demonstrates that the two phenomena are not obligatorily coupled. Podosome disassembly caused by TLR signaling occurs normally in DC lacking MT1-MMP, and instead requires the tumor necrosis factor α–converting enzyme ADAM17 (a disintegrin and metalloprotease 17), which demonstrates a novel role for this “sheddase” in regulating an actin-based structure

Novel Regulatory Mechanisms for Generation of the Soluble Leptin Receptor: Implications for Leptin Action

The adipokine leptin realizes signal transduction via four different membrane-anchored leptin receptor (Ob-R) isoforms in humans. However, the amount of functionally active Ob-R is affected by constitutive shedding of the extracellular domain via a so far unknown mechanism. The product of the cleavage process the so-called soluble leptin receptor (sOb-R) is the main binding protein for leptin in human blood and modulates its bioavailability. sOb-R levels are differentially regulated in metabolic disorders like type 1 diabetes mellitus or obesity and can, therefore, enhance or reduce leptin sensitivity.To describe mechanisms of Ob-R cleavage and to investigate the functional significance of differential sOb-R levels we established a model of HEK293 cells transiently transfected with different human Ob-R isoforms. Using siRNA knockdown experiments we identified ADAM10 (A Disintegrin And Metalloproteinase 10) as a major protease for constitutive and activated Ob-R cleavage. Additionally, the induction of lipotoxicity and apoptosis led to enhanced shedding shown by increased levels of the soluble leptin receptor (sOb-R) in cell supernatants. Conversely, high leptin concentrations and ER stress reduced sOb-R levels. Decreased amounts of sOb-R due to ER stress were accompanied by impaired leptin signaling and reduced leptin binding.Lipotoxicity and apoptosis increased Ob-R cleavage via ADAM10-dependent mechanisms. In contrast high leptin levels and ER stress led to reduced sOb-R levels. While increased sOb-R concentrations seem to directly block leptin action, reduced amounts of sOb-R may reflect decreased membrane expression of Ob-R. These findings could explain changes of leptin sensitivity which are associated with variations of serum sOb-R levels in metabolic diseases

Leptin inhibits sOb-R release from Ob-Rfl or Ob-R219.3 transfected cells into the supernatant.

<p>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.</p

Enhanced release of sOb-R after palmitate incubation is mediated by ADAM10 and ADAM17 and inhibited by oleate.

<p>(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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034787#pone-0034787-g005" target="_blank">Fig. 5</a> D and E, n = 2).</p

Caspase-mediated apoptosis, induced by incubation with staurosporine, activates Ob-R shedding mainly mediated by ADAM10.

<p>(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.</p

ER stress causes downregulation of membrane Ob-R, impaired leptin signaling via Ob-Rfl and decreased sOb-R concentrations.

<p>(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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034787#pone-0034787-g008" target="_blank">Fig. 8</a> A, n = 1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034787#pone-0034787-g008" target="_blank">Fig. 8</a> B, n = 2).</p

Constitutive shedding of human Ob-R isoforms is mainly mediated by ADAM10.

<p>(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.</p

Constitutive shedding of human Ob-R isoforms.

<p>(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.</p

Co-expression of Ob-R219.3 to Ob-Rfl and increased sOb-R levels modulate leptin signaling of Ob-Rfl.

<p>(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.</p