dependent on correct dynamics of vesicle formation and endocytosis. Considering that dynasore inhibits detachment of vesicles but leads to an accumulation of vesicle intermediates which are still connected to the plasma membrane [12], a disturbance of the localization of the receptors and other proteins involved in BMP signal transduction by interference with the endocytic machinery might contribute to the delay and reduction in Smad1/5/8 phosphorylation. However, a direct connection between endocytosis inhibition and altered Smad1/5/8 nuclear translocation dynamics after short treatment with BMP-2 cannot be stated at that point as delayed nuclear translocation might be a consequence of the delayed Smad1/5/8 phosphorylation profile and is not directly linked to altered endocytic processes in the cell. Further experiments on BMP-2 induced Smad1/5/8-BMPRI dissociation in the background of endocytosis inhibition will give more insights in that topic. The BRE-Luc reportergene construct used in this study is derived from the Id1 promoter but an artificial construct which was created to efficiently read out Smad1/5/8 dependent signaling [15]. As it does not display the endogenous full length Id1 promoter, we were interested in the effect of endocytosis inhibition on endogenous BMP-2 induced genes. To analyze global effects of BMP-2 stimulation under endocytosis inhibition conditions, we performed whole genome expression profiling of C2C12 cells. Our data showed 2214 genes that were significantly detected (detection P-value,0.05) and regulated by a cut-off of 1.4 fold (Figure 3A). 39 genes showed exclusive regulation by Dynasore. Among those, genes like cell division cycle 27 (Cdc27) and cyclin G1 (Ccng1) have been shown to be involved in cell cycle regulation [28,29]. Regulation of these genes may be due to the known reversible effects of dynasore on cell growth [12]. 610 genes were commonly regulated by BMP- 2+Dynasore or Dynasore. Among those, genes annotated to pathways such as oxidative phosphorylation, or involved in prostate cancer were regulated. Interestingly, we found genes like Ephrin B1 (Efnb1) or CAAT/enhancer binding protein beta (Cepbp), which are known to be involved in cell migration, transcriptional regulation and control of osteogenic processes [30,31]. We found 925 genes induced by BMP-2+DMSO. Those genes were differently affected by addition of dynasore. In this group we identified two classes of genes, which we defined as target genes dependent or independent of endocytosis (Figure 3B,C). The endocytosis dependence of genes in the first class was evident from the negative effect of dynasore on their expression (expression in the BMP-2+Dynasore treatment as compared to BMP-2+DMSO). Among those, we identified the known BMP-2 target genes Id1, Id3, Dlx2 and Hey1, and genes that could be functionally annotated to Wnt signaling, pathways in cancer or cytokine-cytokine receptor interaction and that have critical roles in osteogenesis. For instance, mitogen-activated protein kinase kinase kinase 7 (Map3k7) was shown to be upstream of p38 and its activation results in promoting Runx2 transcriptional activity and osteoblast differen- tiation [32]. Interleukin 11 (Il11) was shown to positively influence bone formation and is suggested to enhance BMP action in bone [33]. The second class displayed genes that are independent of endocytosis, as judged by the finding that their expression was unaffected by dynasore addition during BMP-2 stimulation. Among those genes, we identified the BMP-2 target genes Id2 and Dlx3, as well as the genes Krt16 and Zbtb2. In addition, we detected the transcription factor zinc finger protein 64 (Zfp64), which was described to be downstream of Runx2 and to be involved in osteogenic differentiation [34]. Moreover, we detected among the endocytosis-independent genes the EGF-like ligand betacellulin (Btc), which influences osteogenesis in MSCs [35]. Spatial Segregation of BMP/Smad Signaling To our knowledge, this is the first report, which shows that BMP-2 induced gene expression is selectively regulated by dynamin-dependent endocytosis; this was achieved by identifying genes whose expression is dependent or independent on endocytic signal transduction. Using qPCR studies, we confirmed those two gene classes, and demonstrated that BMP-2 induced Id1, Id3, Dlx2 and Hey1 gene expression is endocytosis-dependent, whereas Id2, Dlx3, Zbtb2 and Krt16 gene expression is endocytosis-independent (Figure 4A,B). All those genes, except Krt16 and Zbtb2, were identified to be early target genes of BMP signaling and are associated with osteogenic processes [20,21]. Hey1 was shown to be a late early target gene of BMP-2 and knockdown of Hey1 in MSCs resulted in inhibition of osteoblast differentiation [21,36]. Id proteins are antagonists of bHLH transcription factors and are involved in transcriptional regulation. Id1 and Id3 show similar expression patterns during development and double knockout mice die during embryogenesis. Id1 associates with E2A proteins and interferes with the formation of a functional E2A-MyoD heterodimer, negatively influencing myogenesis [16,37]. In contrast, Luan and colleagues reported that Id2 physically interacts with Runx2, inhibiting its osteogenic differen- tiation potential by preventing binding of Runx2 to the OCN promoter [38]. Those studies underline the opposing roles of Id1 and Id2 in differentiation. Interestingly, we could show that Id1 expression was dependent on endocytosis whereas Id2 expression was not (Figure 3, 4A,B). The differential regulation of Id1 and Id2 gene expression was also demonstrated in reportergene assays (Figure 4C). These results suggest that the mode and frequency of endocytosis in C2C12 cells might alter their direction of differentiation (osteogenesis versus myogenesis) by inducing Id1 versus Id2 (Figure 6). Both pathways depend on phosphorylated Smad1/ 5/8; however, the kinetics and site of release from the receptor (endosome or plasma membrane) may determine the fate of activated Smads for further interactions with co-activators or co- repressors, leading to specific recognition of promoter sequences. Interestingly, Dlx2 and Dlx3 are differentially regulated by dynasore treatment, although both belong to the same family of Distal-less homeobox proteins (Figure 3, 4A,B). SiRNA-mediated knockdown of Dlx3 resulted in down-regulation of Runx2 gene expression, highlighting a role for Dlx3 in early stages of osteoblast differentiation [39]. In addition, recent publications reported that miR-31 targets both Dlx3 and Krt16 along with Fgf10, Sclerostin and BAMBI in hair follicles [22,40]. These data underline the similarity in gene expression regulation of Dlx3 and Krt16 as both are identified in the small class of BMP target genes, which are independent of dynamin-controlled endocytosis (Figure 3, 4B). The impact of endocytosis on transcription of target genes has been previously demonstrated for insulin signaling. Expression of the target genes glucokinase or c-fos is specifically initiated by insulin receptor isoform B (IR-B) located at the plasma membrane or in sorting endosomes, respectively [41]. Interestingly c-fos is also regulated by BMP-2 via the endocytosis-dependent Smad pathway (Figure 3B). Endocytosis-independent Smad signaling was shown for activin stimulation, where an activin type I receptor mutant impaired in endocytic uptake was still able to induce Smad2 phosphorylation and subsequent transcriptional activation [42]. Activation of the TGFb mediated Smad2/3 pathway was proposed to include presentation of Smads via Smad anchor for receptor activation (SARA), clathrin-mediated endocytosis of activated receptors and release from the receptors in early endosomes [9]. However, there are controversial reports on the necessity of clathrin-mediated endocytosis of phosphorylated Smad2 to induce transcription [43,44]. Another endosomal protein, endofin, was found to positively influence TGFb signaling [45,46]. PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e25163