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 6 SD of n$3 experiments. doi:10.1371/journal.pone.0034787.g007 and determination of ER stress markers, respectively. Cell viability did not change significantly over the 24 h of tunicamycin incubation determined by CCK-8 assay (data not shown). Fig. 8A illustrates the successful activation of ER-stress markers ‘‘immunoglobulin heavy chain binding protein’’ (BiP), also referred as 78-kDa glucose- regulated protein (GRP78) and the C/EBP homology protein (CHOP). Another marker protein named protein-disulfide- isomerase (PDI) remained unaffected. 125I-leptin binding assay revealed a 60% decreased leptin binding after 4 h of tunicamycin incubation which further declined to 15% of control level after 24 h (Fig. 8 B). Leptin-induced STAT3 phosphorylation was decreased by 47% (P,0.01) after 4 h and completely blocked after 12 h incubation with tunicamycin (Fig. 8 C). Total STAT3 amounts remained unchanged after tunicamycin treatment (Fig. 8 D). To elucidate if the reduced membrane Ob-R expression is reflected by decreased sOb-R levels, we incubated Ob-Rfl as well as Ob-R219.3 transfected cells with tunicamycin (3–20 mg/ml) for 24–48 h. Fig. 8E shows that tunicamycin incubation led to 65% (P,0.001) decreased sOb-R levels compared to the non-treated sample already after 24 h. The inhibition reached a maximum of 92% and 90% for Ob- Rfl and Ob-R219.3 after 48 h. Discussion In previous studies we and others showed that sOb-R represents the main leptin binding activity in human blood [15] and modulates the bioavailability of free leptin [15–19]. Detailed knowledge about the basic shedding mechanisms of Ob-R is mandatory to manipulate leptin actions by increasing or decreasing sOb-R levels in conditions of leptin deficiency or resistance. In the present study we elucidated regulatory mechanisms for sOb-R generation covering receptor shedding and receptor trafficking in HEK cells with a transient overexpres- sion of different Ob-R isoforms. We evidenced that the four known human membrane-anchored Ob-R isoforms were sub- strates for generation of sOb-R with constitutively low levels. The degree to which each isoform contributes to the pool of serum sOb-R appeared to correlate with the respective Ob-R total and membrane protein expression and seemed to be inversely associated with the length of the cytoplasmic domain. Previously, Ob-Rfl was demonstrated to be the main signal-transducing isoform [11] and Ob-R219.3 was shown to be expressed Regulatory Mechanisms for sOb-R Generation abundantly in peripheral tissues and displayed the highest shedding rate in our in-vitro experiments. Therefore, we focused our investigations in the present study on these two physiologically relevant Ob-R-splice variants. ADAM10 and or ADAM17 are known members of the metalloprotease family that cleave transmembrane receptors like IL-6R, IL-15Ra and the growth hormone receptor which like Ob-R belong to the class I cytokine receptor family [37–39]. Downregulation of sOb-R by ADAM10 or ADAM17 inhibitors and through ADAM specific siRNA knockdown identified ADAM10 as the major protease for constitutive Ob-R shedding. ADAM17 appeared to be involved in the constitutive cleavage process only to a minor degree. This hypothesis was further supported by measurements of sOb-R levels in serum of ADAM17ex/ex mice (data not shown) [43]. Serum sOb-R concentrations of these mice did not differ significantly from those of wild type mice despite of the protease knockdown. We further clarified mechanisms that induce or reduce the cleavage of membrane Ob-R. The detected increase of Ob-R shedding upon PKC activation is in line with previously published results that showed an activating effect of PKC stimulation on Ob-R cleavage [13,14]. Interestingly, our data point to an isoform-dependent differential regulation of the shedding process: while ADAM10 appears to be responsible for PMA-induced shedding of Ob-R219.3, Ob-Rfl was shed by ADAM10 as well as ADAM17 in response to PMA treatment. As we could not completely inhibit constitutive and PKC-activated Ob-R shedding by knocking down ADAM10 and ADAM17 in our cell culture experiments, effects of other proteases cannot be fully excluded. Recently, Wauman et al. [44] showed that ADAM10 and ADAM17 were involved in RNF41 induced shedding of the human long Ob-R isoform and mouse short Ob-R isoform which confirms our findings that especially these two ADAM family members play an important role in activated Ob-R ectodomain-shedding. Additionally, we demonstrated that apoptosis induced by staurosporine and doxorubicin (Supporting information Fig. S5) activated shedding from Ob-R219.3 trans- fected cells. In contrast, the shedding rate of Ob-Rfl transfected cells remained negligible with both substances. This is interesting because it is in contrast to PMA incubation which increased shedding of Ob-Rfl and Ob-R219.3 to approximately the same extent. These results suggest that the amount of shed Ob-R depends on the shedding stimulus and the isoform-specific length of the cytoplasmic domain. Perhaps turnover of Ob-Rfl is greater than of Ob-R219.3, which would result in a bigger abundance of the short isoform Ob-R219.3 compared to the long isoform Ob- Rfl. A similar effect had been described for the human growth hormone receptor [45]. In addition, the longer intracellular domain of Ob-Rfl could also have a stabilizing effect and therefore protects Ob-Rfl from shedding. Differences in shedding efficacy between the short isoform Ob-R219.3 and the long isoform Ob- Rfl might be of importance for leptin actions. One supposed example for the physiological relevance of this process is leptin- transport across the blood brain barrier which is suggested to be dependent on the short isoforms [46,47]. The preferential shedding of short isoforms could restrain this transport leading to impaired central leptin actions without affecting its peripheral effects. Moreover, in peripheral tissues with high expression of short Ob-R isoforms increased shedding could have a dual effect on leptin signaling via Ob-Rfl. Shedding of preferably short Ob-R isoforms could make the cells more sensitive to leptin. But with increasing levels of generated sOb-R an inhibitory effect of the released Ob-R ectodomain on leptin signaling may prevail. By use of the broad spectrum caspase inhibitor Z-VAD(OMe)-FMK we proved that increased shedding due to staurosporine treatment PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34787