Spatial Segregation of BMP/Smad Signaling Figure 4. Validation of two classes of genes, which are dependent or independent of endocytosis. (A,B) Serum-starved C2C12 cells were treated for 2 h with 40 mM dynasore or 0.05% DMSO prior to stimulation with 30 nM BMP-2 for 6 h in medium containing dynasore or DMSO. Total RNA was isolated, cDNA was synthesized and mRNA expression was analyzed by qPCR using specific mouse primers (Table S1). Histograms show mean normalized expression (MNE) with standard error of duplicate measurements relative to the housekeeping gene HPRT. The analysis shown is representative of three independent experiments. (C) C2C12 cells were co-transfected with Id1-Luc or Id2-Luc and RL-TK. Cells were serum-starved and treated with 40 mM dynasore or 0.05% DMSO for 1 h prior to stimulation with 3 nM BMP-2 for 24 h in medium containing dynasore or DMSO. Relative luciferase activity (RLA) of Id1- or Id2-driven luciferase compared to constitutive expression of RL-TK is shown. Results are shown as mean 6 s.d. of triplicate measurements, representative of three independent experiments. doi:10.1371/journal.pone.0025163.g004 Osteoblast differentiation of C2C12 cells is arrested by short-term inhibition of dynamin-dependent endocytosis BMPs are potent inducers of mesenchymal cells to differentiate into osteoblasts and chondroblasts [5]. The C2C12 precursor cell line provides a biologically suitable model to investigate the BMP-2- induced transcriptional cascade which initiates osteoblast differen- tiation [24]. We have previously shown that ALP production is dependent on Smad and non-Smad signaling, as well as on both clathrin- and caveolae-mediated endocytosis [6,11]. Considering that inhibition of dynamin-dependent endocytosis affects Smad1/ 5/8 signaling kinetics and BMP-2-induced transcriptional expres- sion profiles, we examined the consequences of these altered signaling properties on osteoblast differentiation. Differentiation was analyzed after 72 h of BMP-2 administration to serum-starved C2C12 cells, since the expression of osteoblast markers such as alkaline phosphatase (ALP), osteocalcin (OCN) and osteopontin (OPN) is detectable after this time period [24]. Application of dynasore for 2 h efficiently blocked endocytic uptake in C2C12 cells in our study and this effect was reversed by wash-out of the inhibitor [12]. However, long-term treatment over several days has lead to cell death. Therefore, dynasore was added only during starvation and the initial 4 h of BMP-2 stimulation in the differentiation assays. To test whether inhibition of endocytosis during the initial phase of BMP-2 signal transduction affects osteoblast differentiation, ALP assays were performed (Figure 5A). Application of dynasore during the first 4 h of BMP-2 stimulation significantly reduced ALP activity, even though the ligand was continuously present during the following 68 h. Application of the ligand for these additional 68 h did not circumvent the impaired ALP activity. The importance of the initial phase of stimulation was also demonstrated by ALP assays after 72 h, where BMP-2 was applied only during the initial 4 h (Figure 5B). These results suggest that correct signaling properties in the initial phase of BMP-2 stimulation are important for commitment of the cells towards the osteoblast lineage. To gain more insight into the effect of inhibition of dynamin- dependent endocytosis in osteoblast differentiation, we performed qPCR analysis for osteoblast markers. Cells were pre-treated with dynasore and subsequently stimulated with BMP-2 for 72 h. During the initial 4 h of BMP-2 stimulation, dynasore was added. The expression analysis of osteoblast markers like ALP, OCN and OPN revealed that dynasore treatment results in down-regulation of these genes (Figure 5C–E). Interestingly, combining BMP-2 with dynasore resulted in higher and sustained expression levels of crucial early markers for osteoblast differentiation like runt related transcription factor 2 (Runx2) and osterix (Osx) relative to control cells (Figure 5F,G). Taken together, the blockade of dynamin- dependent endocytosis during the initial phase of BMP-2 application resulted in down-regulation of osteoblast markers, thereby attenuating osteoblast differentiation. In contrast, the expression levels of early markers such as Runx2 and Osx were increased by dynasore treatment. Discussion Fine-tuning of signaling events is essential for living cells to regulate proper signal conversion and outcome. Endocytosis and PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e25163