Regulatory Mechanisms for sOb-R Generation 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 mM) 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 6 SD of n$3 experiments. doi:10.1371/journal.pone.0034787.g003 increase of sOb-R by up to 3%(P,0.05) (Fig. 4 B and C). To clarify if ADAM10 and ADAM17 are involved in staurosporine- induced shedding we knocked down ADAM10 or ADAM17 with a specific siRNA as described before and incubated Ob-R219.3 transfected cells with staurosporine 0.5 mM for 48 h. Fig. 4D shows that ADAM10 siRNA knockdown resulted in 84% (P,0.01) whereas a knockdown of ADAM17 diminished sOb-R levels by 23% (p = 0.26) compared to the sample transfected with a non- targeting siRNA. To elucidate if the effects on sOb-R generation were staurosporine-specific we incubated Ob-R transfected HEK cells with doxorubicin (inhibitor of topoisomerase II) as a more specific inducer of apoptosis. We demonstrated that doxorubicin increased the amount of shed Ob-R219.3 in a comparable manner as 0.1 mM Staurosporine, whereas no significant changes could be determined for Ob-Rfl (Supporting information Fig. S5). Lipotoxicity and apoptosis induced by the saturated FFA palmitate increase sOb-R levels. Lipotoxicity caused by elevated free fatty acids in tissues other than the adipose tissue could finally cause cell death through apoptosis. So we speculated that free fatty acids influence Ob-R cleaveage. By incubating Ob-R219.3 or Ob-Rfl transfected HEK cells with oleate or palmitate (0.5 mM–1 mM) we could show that palmitate but not oleate increased the amount of shed Ob-R. Ob-R219.3 transfected cells showed up to 3.5-fold (P,0.01) and Ob-Rfl transfected cells up to 1.4-fold(P,0.05) elevated sOb-R levels in the supernatant after 48 h (Fig. 5 A). To elucidate the role of ADAM10 and ADAM17 in palmitate- mediated shedding we knocked down ADAM10 or ADAM17 with a specific siRNA as described before and incubated Ob-R219.3 or Ob-Rfl transfected cells with palmitate 1 mM for 48 h. Fig. 5 B displays that knockdown of ADAM10 resulted in up to 81% (Ob- R219.3, P,0.01) or 87% (Ob-Rfl, P,0.05) decreased amounts of sOb-R whereas a knockdown of ADAM17 diminished sOb-R levels by 44% (Ob-R219.3, P,0.05) or 11% (Ob-Rfl, P = 0.11) compared to cells treated with a non-targeting siRNA. The observed increase was accompanied by decreased cell viability measured by CCK-8 assay (data not shown) as a consequence of apoptosis demonstrated through detection of cleaved caspase-3 and cleaved PARP (Fig. 5 C and E). Interestingly a co-incubation of Ob-R-transfected cells with palmitate 1 mM and broad spectrum caspase inhibitor Z-VAD(OMe)-FMK (100 mM) inhibited induction of caspase-3 and PARP-cleavage but did neither restore cell viability (data not shown) nor decreased sOb-R to basal levels (Fig. 5 D and E). In contrast co-incubation of Ob-R transfected cells with palmitate 0.5–1 mM and increasing concentrations of oleate 0.1–0.5 mM reversed the lipotoxic effects of palmitate (Fig. 5F–H) and decereased sOb-R concentration to basal levels (Fig. 5F and G). Modulation of leptin-mediated STAT3 phosphorylation by sOb-R and Ob-R isoform 219.3 We elucidated if leptin actions via STAT3 phosphorylation might be modulated by co-expression of Ob-R219.3. Ob-Rfl and PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34787