like inhibitory Smad7 affect the signal transduction into the nucleus and thereby modulate the signal propagation, resulting in transcriptional activation or repression [2]. Endocytosis of transmembrane receptors is an important mechanism to control receptor availability at the cell surface or to induce attenuation of signal transduction by sequestering receptors from modulators [9]. A well-described mechanism for cellular uptake is clathrin-mediated endocytosis. Thereby, cla- thrin-coated pits pinch off the membrane in a dynamin-dependent manner. Internalized receptors are either sorted for recycling to the plasma membrane, are degraded in lysosomes or can use the endosome as a signaling platform, where downstream components are presented for further activation. Another mode of internali- zation is uptake via caveolae-mediated endocytosis; this non- clathrin endocytic pathway uses membrane invaginations contain- ing the membrane protein caveolin, and also requires dynamin for the pinching off of caveolar vesicles [10]. BMP receptors can enter the cell by different endocytic routes. Different intracellular cascades are initiated dependent on receptor localization in distinct compartments of the plasma membrane and their oligomerization mode [11]. Phosphorylation of Smad1/5/8 by BMPRI is induced at the plasma membrane, but the continuation of the Smad1/5/8 dependent pathway which results in transcriptional activation of specific target genes was shown to rely on clathrin-mediated endocytosis, while the Smad- independent pathway required both clathrin- and caveolae- mediated endocytosis to exert transcriptional activity [11]. In this study, we further investigated the dependence of the transcriptional regulation in BMP signaling on endocytosis and the consequences of endocytosis inhibition on osteoblast differentia- tion of C2C12 cells. By the use of dynasore, an inhibitor of dynamin-dependent endocytosis [12], we show that the kinetics of BMP-2 induced Smad1/5/8 phosphorylation and nuclear trans- location are delayed and Smad1/5/8 phosphorylation is reduced. Although Smad1/5/8 phosphorylation and nuclear translocation still occur, the delay in the initial kinetics results in down- regulation of the BMP responsive element (BRE) reportergene activity and osteoblast differentiation. To provide more insight on Smad-dependent transcriptional activation, we performed a whole genome microarray analysis and identified two classes of BMP-2 induced genes, which display differential sensitivity to inhibition of dynamin-dependent endocytosis. Thereby, we could cluster genes into endocytosis dependent and endocytosis independent groups. Our data highlight the importance of endocytosis for transmission of an extracellular BMP-2 signal into the nucleus to activate a transcriptional gene network involved in osteoblast differentiation. Additionally, we suggest a novel Smad signal transduction cascade induced by BMP-2, which does not depend on endocytosis. Results The kinetics of Smad1/5/8 phosphorylation is influenced by dynamin-dependent endocytosis BMP signaling is regulated by receptor oligomerization and membrane localization and its subsequent intracellular signal propagation is controlled by different endocytic routes [6,11]. To gain further insights into the endocytic regulation of BMP signaling pathways, we used the small molecule inhibitor dynasore, which specifically interferes with dynamin-dependent endocytosis by reversibly blocking the GTPase activity of dynamin. Dynamin is crucial in vesicle fission prior to the release of vesicles from the plasma membrane [13]. Macia and colleagues showed that the inhibitor dynasore does not abolish vesicle formation but pinching off the membrane invaginations is blocked [12]. Spatial Segregation of BMP/Smad Signaling To test the efficiency of dynasore ability to inhibit endocytosis in our system, C2C12 cells were subjected to fluorescent transferrin uptake to monitor clathrin-mediated endocytosis [14]. C2C12 cells incubated with Alexa594-transferrin or DMSO as control showed intracellular transferrin staining after 15 min (Figure 1A, left panels). In contrast, transferrin uptake was potently inhibited by treating the cells for 2 h with 40 mM dynasore (Figure 1A, right panels). The quantification of this experiment underlines the potential of dynasore to efficiently inhibit endocytosis in C2C12 cells (Figure 1B). In all subsequent assays transferrin uptake was measured in parallel to validate the treatment conditions with dynasore (data not shown). We have previously shown that Smad1/5/8 phosphorylation is induced at the plasma membrane and it is not affected by treating cells with specific inhibitors for caveolae- or clathrin-dependent endocytosis [11]. To extend these findings, we now analyzed the effects of dynasore on Smad1/5/8 phosphorylation in a time- dependent manner (Figure 1C). C2C12 cells pre-treated with dynasore were stimulated for varying periods with BMP-2, followed by Western blotting with an antibody to C-terminally phosphorylated Smad1/5/8 (pSmad1/5/8). In control cells, phosphorylation of Smad1/5/8 was rapidly induced by BMP-2 after 5 min, peaked at 10 min, and stayed at sustained levels for up to 60 min (Figure 1C, lanes 1–5). The Smad1/5/8 phosphoryla- tion kinetics was delayed and the levels were reduced in cells treated with dynasore. In the presence of dynasore, phosphory- lation of Smad1/5/8 was much lower after 5 min to 10 min and became clearly apparent after 30 min to 60 min (Figure 1C, lanes 6–10). This result confirms that phosphorylation of Smad1/5/8 Figure 1. Phosphorylation of Smad1/5/8 is delayed by inhibition of dynamin-dependent endocytosis. (A) Serum starved C2C12 cells were treated for 2 h with 40 mM dynasore or 0.05% DMSO and incubated for 15 min at 37uC with Alexa594-transferrin in the presence of dynasore and DMSO. Cells were fixed and transferrin uptake was monitored by fluorescence microscopy. Lower panels show Hoechst staining of the respective cells. Bar, 10 mm. (B) Quantification of transferrin uptake shown in (A). The histogram depicts the amount of internalized transferrin, derived from the total fluorescence signal of Alexa594-transferrin within the cell boundaries. The results are mean 6 s.d. of at least 400 cells. AU, arbitrary units. (C) Serum-starved C2C12 cells were treated for 2 h with 40 mM dynasore or 0.05% DSMO prior to stimulation with 10 nM BMP-2 for the indicated time periods in medium containing dynasore or DMSO. Samples were processed for immuno- blotting with anti-phospho-specific Smad1/5/8 antibody (anti-pSmad1/ 5/8) or GAPDH antibody (anti-GAPDH) as loading control. The Western Blot shown is representative of three independent experiments. doi:10.1371/journal.pone.0025163.g001 PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e25163