Figure 1. Extracellular Bmper is actively internal- ized by selective cell types. (a) MECs were treated with 6 nM of puriﬁed, cleaved full-length mouse recombinant Bmper at temperatures that promote (37°C) and inhibit (4°C) active transport. (b) Bmper is internalized by selective cell types, including embryonic endothelial cells (C166), HCAECs, rat neonatal ventricular cardiomyocytes (RVC), and MEFs, but not by HEK-293 or COS7 cells. Both the N- and C-terminal fragments of Bmper are presented in the Western analysis in panels a and b, whereas for clarity the N-terminal frag- ment only is presented in the Western analysis of all subsequent panels. (c–i) Fluorescence confocal microscopy was used to examine the intracellular localization of Bmper. MECs were treated with re- combinant Bmper (6 nM). Panels c, d, and e rep- resent images of bright ﬁeld, IgG control (FITC), and nuclear staining with DAPI (blue), respectively. (f) Endogenous Bmper (DAPI overlay) was de- tected in MECs in a distinct punctuate perinuclear pattern that might represent the secreted form of Bmper. (g) When treated with recombinant Bmper for 5 min, a signiﬁcant amount of Bmper was de- tected on the cell surface. (h) Bmper accumulated in large punctuate vesicles in MECs treated with Bmper and chased with media containing chlo- roquine. The excitation power of FITC had to be reduced by 50% to prevent the pixel saturation of ﬂuorescent Bmper in the presence of chloroquine. (i) Graphical representation of the FITC intensity per unit area of cells treated with a pulse of exog- enous Bmper and then chased with chloroquine. Fluorescent intensity is relative to cells treated with Bmper and stained with an isotype (IgG) control. The differences in the effects of endogenous Bm- per, rBmper pulse, and rBmper pulse followed by chloroquine chase were statistically signiﬁcant (*, P < 0.05) from one another and are represented by the bar in panel i. Samples were analyzed for statistics as described in Materials and methods. that this may be a broadly applied but selective mechanism for regulating Bmp signaling. Results Endocytic internalization of extracellular Bmper While probing for the effects of Bmper on Bmp6-mediated Cox2 induction (Ren et al., 2007), we discovered that recombi- nant Bmper (6 nM) was internalized to the cytosol when added to the culture media of endothelial cells (Fig. 1 a). As a first step to determine whether this observation represented a biologi- cally relevant phenomenon of internalization, we answered the following questions: does Bmper cross the membrane of endo- thelial cells and, if so, is this through passive absorption or active transport? Is this phenomenon selective to certain cell types? Which intracellular pathway does Bmper traffic through? What are the structural requirements of Bmper protein that me- diate internalization? How does Bmper internalization affect Bmp signaling? The mouse embryo-derived endothelial cell line (mouse endothelial cell [MEC]) expresses all of the components re- quired for appropriate Bmp signaling (Valdimarsdottir et al., 2002). To assess whether Bmper internalizes by an active pro- cess or through passive flow, we compared MECs treated with 6 nM of recombinant Bmper at temperatures that promote (37°C) and inhibit (4°C) active transport (Fig. 1 a). Bmper is processed into two fragments that remain associated by disulfide bonds (Binnerts et al., 2004; Rentzsch et al., 2006); both the N- and C- terminal fragments of Bmper were detected by Western analysis 598 JCB • VOLUME 184 • NUMBER 4 • 2009