Figure 6. Bmper regulates Bmp4 activity in a concentration-dependent manner. (a) A rep- resentative Western blot (n = 3) of a dose– response experiment using MECs treated over a range of Bmper concentrations (0–100 nM) while holding Bmp4 constant (1 nM). Noggin was used as a positive control for inhibiting Bmp4 activity. The N-terminal fragment of Bmper is presented for the Western analysis. (b) The difference between the effects of Bmper (squares) and Noggin (triangles) on Bmp4 activ- ity in the range of 0.1 to 5 nM was statistically signiﬁcant (*, P < 0.05). (c) Representative Western blot showing the effects of a low (0.5 nM) and high (50 nM) molar concentra- tion of Bmper on 1.0 nM of Bmp4-mediated cleaved Caspase-3 activity as a readout for apoptosis in primary day 15.5 lung MEFs. (d) Bar Graph showing the quantity of cleaved Caspase-3 activity from panel c. Top bar rep- resents signiﬁcant statistical difference from the untreated samples (*, P < 0.05). The dif- ferences in the effects of low versus high molar concentrations of Bmper on Bmp4-attenuated apoptosis were statistically signiﬁcant (*, P < 0.05) and are represented by the bottom bar in panel d. Samples were analyzed for statis- tics as described in Materials and methods. further during trafficking using Rab7, a GTPase that marks the late endosome compartment (Bucci et al., 2000; Fig. 5 c). Even at this late stage in vesicular transport, Bmper and Bmp4 re- mained intimately colocalized within perinuclear Rab7-express- ing endosomes. The physical proximity and functional role of Rab7 in lysosomal degradation provides an additional layer of evidence that Bmper targets Bmp4 for degradation to restrict its signaling at the cellular level and is consistent with findings that a Rab7-dependent endocytic transport mechanism targets Dpp for degradation to establish a long range Dpp concentration gra- dient during Drosophila development (Entchev et al., 2000). Bmper regulates Bmp4 activity in a concentration-dependent manner Our discovery of a regulatory role for Bmper to regulate Bmp signaling within the endocytic pathway provides an elegant ex- planation for the inhibitory effects of Bmper on Bmp activity identified by multiple laboratories (Moser et al., 2003; Binnerts et al., 2004; Coles et al., 2004; Rentzsch et al., 2006; Zhang et al., 2007), but fails to reconcile other studies that indicate pro-Bmp activities of Bmper (Conley et al., 2000; Coles et al., 2004; Kamimura et al., 2004; Ikeya et al., 2006; Rentzsch et al., 2006). A recently reported concentration-dependent model helps ex- plain both anti- and pro-Bmp activity by showing that Drosophila Cv-2 stimulates Bmp signaling at low concentrations and inhibits Bmp signaling at high concentrations (Serpe et al., 2008). We reasoned that a threshold effect for Bmper-dependent Bmp inter- nalization would explain its opposing activities on canonical Bmp signaling. We tested a range of Bmper concentrations (0–100 nM) on Bmp4-dependent Smad activity and Bmp4 endo- cytosis, using the well characterized Bmp inhibitor Noggin as a control (Fig. 6, a and b). At molar concentrations below those of Bmp4, Bmper enhanced Smad activation, whereas at molar con- centrations higher than Bmp4, Bmper attenuated Bmp4-mediated Smad activation and simultaneously triggered Bmp4 endocytosis (Fig. 6, a and b). Binding assays indicate that Bmper binds to Bmp2 with a 2:1 molar stoichiometry (Zhang et al., 2007), and remarkably it was when this molar ratio was exceeded that Bmper restricted Bmp4 signaling and triggered endocytosis of the Bmp4–Bmper complex. We also found that Noggin triggered Bmp4 endocytosis at concentrations that exceeded stoichiomet- ric equivalence with Bmp4 (Fig. 6 a). The kinetics of Bmper- and Noggin-dependent Bmp4 internalization were similar, yet only Bmper increased Bmp4-dependent Smad signaling at below threshold concentrations. The selectivity of this endocytic mech- anism for other secreted modulators of Bmp signaling is further demonstrated in the results describing Fig. 8. We explored the concentration-dependent relationship of Bmper with Bmp4 in a biologically relevant model of Bmp sig- naling. At day 15.5 of embryonic lung development, the lung mesenchyme begins to regress through a Bmp4-dependent mechanism as the terminal epithelium expands into the distal airways (Bellusci et al., 1996). We found Bmper to be expressed in the supporting mesenchyme of the lung (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200808064/DC1), which is consistent with a previous study (Ikeya et al., 2006). Bmp4, in contrast, is expressed in the distal tips of the lung epithelium and at lower levels in the adjacent lung mesenchyme (Bellusci et al., 1996). If a concentration-dependent model for regulating Bmp signaling by Bmper is correct, then Bmper should also regulate Bmp4-mediated survival of the embryonic lung mesenchyme in a concentration-dependent manner. To test this theory, we treated ENDOCYTIC REGULATION OF BMP SIGNALING BY BMPER • Kelley et al. 603