Spatial Segregation of BMP/Smad Signaling Figure 6. Hypothetical model. Id1 prevents myogenic differentiation by interfering with the transcriptional activity of E2A/MyoD complexes [16]. During early stages of osteoblast differentiation, Runx2 is expressed. Expression levels of Runx2 need to be tightly controlled during later stages of differentiation, as Runx2 overexpression prevents late osteoblast differentiation [52]. In addition, it was demonstrated that Id2 prevents Runx2 binding to the late osteoblast marker promoter of OCN [38]. We suggest that differential gene expression regulation reliant on endocytic signal transduction provides an additional mechanism for the cell to regulate differentiation processes. doi:10.1371/journal.pone.0025163.g006 The association of the TGFb and BMP receptors with specific adaptor proteins for clathrin-mediated endocytosis might be important to trigger this step in time resolved signal transduction. For the BMP receptor type II (BMPRII), interaction with clathrin adaptor proteins Eps15R and AP2 has been demonstrated [11,47]. Spatial segregation of Smad signaling as shown here by altered phosphorylation dynamics might be triggered through this initial step of adaptor protein interaction allowing or excluding the receptors to internalize via dynamin dependent endocytosis events. However, because dynasore imparts a general blockade of endocytosis, it is possible that the inhibition of signaling by dynasore is due to a requirement for endocytosis or specific intracellular localization of another downstream component in the pathway. In order to resolve both segregated Smad pathways in more detail, specific Smad-interactome studies by concomitant BMP-2 stimulation and endocytosis inhibition would give more insights into the selective regulation. The physiological importance of our findings is highlighted in osteoblastic differentiation studies. We show that dynasore treatment during the initial 4 h of BMP-2 induced differentiation inhibited ALP activity, which goes hand in hand with the down- regulation of the osteoblast marker genes ALP, OCN and OPN in qPCR analysis (Figure 5A, C–E). Interestingly, the crucial early marker genes Runx2 and Osx [48,49] were up-regulated (Figure 5F,G). The importance of concise Runx2 expression levels at multiple stages during differentiation processes has been extensively reviewed [50,51]. In addition to this, Liu and colleagues reported that over-expression of Runx2 inhibited late stages of osteoblast differentiation [52]. The requirement of a confined, spatiotemporal expression pattern as described for Runx2 could also be demonstrated for Osx. The forced expression of Osx in different cell types was incapable of inducing osteoblast differentiation [53,54]. Our observations suggest that interference with the BMP-2-induced transcriptional cascade by dynasore might lead to the entrapment of cells in an early osteoblast stage, which cannot progress towards late osteoblast differentiation due to disturbed expression of important early osteoblast markers Runx2 and Osx. Our results indicate that negative Runx2 regulators are induced via the endocytosis-dependent BMP-2 signaling pathway. The transcription factor E4BP4 and the homeodomain protein Nkx3.2 were already described as negative regulators of Runx2 expression [55,56]. Id2 inhibits Runx2 activity by physical interaction, which prevents Runx2 binding to the OCN promoter [38]. Since we analyzed gene expression on a whole-genome level after 6 h of BMP-2 stimulation, and Runx2 down-regulation is needed during late stages of osteoblast differentiation (24 h [51]), further analysis during the complete differentiation process will help to understand the underlying mechanism and to identify all crucial players. The importance of the initial stimulation phase in BMP-induced differentiation was described by van Bezooijen and colleagues, who reported that BMPs need to be present for 24 h on pre- osteoblast KS483 cells to induce osteoblast differentiation [57]. In line with this notion, we demonstrated that application of BMP-2 for a minimum of 4 h is sufficient to induce osteoblast differentiation in C2C12 cells (Figure 5B). In summary, we show here for the first time that BMP/Smad signaling is spatially segregated into the predominant dynamin- dependent endocytosis path and a second pathway, which is endocytosis-independent. These pathways differ in their kinetics and in the regulation of distinct target genes, thus allowing for differential regulation of members within the Id and Dlx family. Functional interference with the dynamin-dependent pathway in the initial phase of osteoblastic differentiation arrests precursor cells, suggesting that the early stages of differentiation are strongly dependent on this route of the Smad pathway. Furthermore, we emphasize that treatment with BMP-2 for a limited time is sufficient to promote the osteoblast differentiation program. Materials and Methods Cell culture, transfection and materials C2C12 mouse mesenchymal precursor cells (ATCC) were grown at 37uC with 10% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) (Biochrom) supplemented with 10% (v/v) Fetal Calf Serum (FCS), 1 mM Glutamine, Penicillin (100 units/ml) and Streptomycin (10 mg/ml) (PAA). Cells were transfected using PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e25163