Regulatory Mechanisms for sOb-R Generation 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 (37uC, 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 (37uC, 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 6 SD of n$3 experiments; n.d., not detectable. doi:10.1371/journal.pone.0034787.g001 with a 100-fold higher specificity towards ADAM10 than to other proteases or GW280264X (GW) that blocks both ADAM10 and ADAM17 with comparable IC50 values [40,41]. Fig. 2 A reveals that GI diminished constitutive shedding by 53% (P,0.01) whereas incubation with GW resulted in 52% (P,0.01) decreased sOb-R levels. To further verify to which extent the different ADAMs participate in Ob-R cleavage of the two selected isoforms, we next performed ADAM10 and ADAM17 siRNA knockdown experiments. HEK cells were first transfected with an ADAM10 or ADAM17 specific siRNA or a non-targeting siRNA. 24 hours later the siRNA transfected cells were transfected with Ob-Rfl or Ob-R219.3. Quantitative RT-PCR measurements revealed a knockdown of ADAM10 or ADAM17 mRNA expression of at least 60% (P,0.001) 24 h–72 h post transfection (Fig. 2 B). The observed mRNA knockdown was paralleled by a decrease in ADAM10 and ADAM17 protein levels (Fig. 2 C). ADAM10 knockdown sufficiently blocked the constitutive release of sOb-R into the supernatant of Ob-Rfl transfected cells by up to 57% (Fig. 2 D, P,0.001) whereas ADAM17 knockdown less effectively diminished sOb-R levels up to 30% (P,0.01). The release of sOb-R by Ob-R219.3 transfected cells was decreased with a maximum of 64% (Fig. 2 E P,0.001) after ADAM10 but only up to 39% (P,0.01) by ADAM17 siRNA-mediated knockdown (Fig. 2 E). Induction of Ob-R shedding PKC stimulation increases Ob-R shedding. It is known that metalloprotease-mediated shedding processes can be induced by activation of PKC [42]. Hence we transfected HEK cells transiently with Ob-Rfl or Ob-R219.3 and stimulated these cells with the PKC-activator PMA (10 ng/ml) for 24 h. The incubation resulted in a 3.98-fold (P,0.01)/4.14-fold (P,0.001) increase of sOb-R in the cell supernatant (Fig. 3 A). Co-incubation of Ob- R219.3 transfected cells with the protein kinase inhibitor staurosporine (0.01 mM) inhibited the PMA stimulated increase of sOb-R concentration after 24 h by 60% (P,0.05) (Fig. 3 B). PKC-induced Ob-R shedding is mediated by ADAM10 and to a minor degree by ADAM17. To elucidate the importance of ADAM10 and ADAM17 for PKC-induced Ob-R shedding, Ob-R219.3 transfected cells were incubated with the specific ADAM10 or ADAM10/17 inhibitors GI and GW for 24 h. Results shown in supporting information Fig. S3 reveal that GI diminished PMA-induced shedding by 48% (P,0.01) and that GW-mediated decrease was 51% (P,0.01). To clarify the effect of either ADAM on PKC-induced Ob-R shedding, we knocked down ADAM10 and ADAM17 in Ob-Rfl or Ob-R219.3 transfected HEK cells. PMA-induced shedding of Ob-Rfl transfected cells was only decreased by up to 20% (P,0.05) independently of the siRNA applied (Fig. 3 C). However simultaneous knockdown of ADAM10 and ADAM17 led to a significant decrease of 43% (P,0.01) supporting that both ADAMs participate in PKC-induced shedding of Ob-Rfl. PMA- induced elevation of sOb-R in the supernatant of Ob-R219.3 transfected cells was dimished by 52% (P,0.01) after knocking down ADAM10 (Fig. 3 D) compared to the sample treated with non-targeting siRNA. Knockdown of ADAM17 only decreased sOb-R concentration by 24% (P,0.001). Caspase-mediated apoptosis increases Ob-R shedding dependent on ADAM10 expression. Staurosporine, beside its inhibitory effect on protein kinases is known to be an effective inducer of apoptosis. To investigate a potential association between apoptosis and Ob-R shedding we incubated Ob-R219.3 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34787