Spatial Segregation of BMP/Smad Signaling Figure 5. Osteoblast differentiation is arrested by inhibition of dynamin-dependent endocytosis. (A,B) Confluent C2C12 cells were serum-starved for 2 h prior to stimulation with the indicated concentrations of BMP-2. After 72 h of stimulation, cells were lysed and ALP activity was measured at 405 nm by conversion of para-nitrophenylphosphate. A schematic representation of the treatment is depicted above the respective histogram. The histograms show mean 6 s.d. of triplicate measurements representative of three independent experiments. (A) During starvation and initial 4 h of stimulation, 40 mM dynasore or 0.05% DSMO were added. (P-values in relation to control treated samples). (B) After 4 h of stimulation, BMP-2-containing medium was replaced by medium without BMP-2. (** P-values in relation to unstimulated samples; 30 nM: P = 0.008, 60 nM: P = 0.0017) (C–G) Confluent C2C12 cells were treated as described in (A) with 30 nM BMP-2. 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 GAPDH. The results are representative of three independent experiments. doi:10.1371/journal.pone.0025163.g005 recycling of membrane-associated proteins, including (but not limited to) receptor complexes and other signaling proteins, provides an important mechanism that regulates signal transduc- tion. Moreover, endosomes derived from the plasma membrane can serve as signaling platforms for a number of signal transduction cascades, and certain signaling components are exclusively localized to endosomes [10]. The role of endocytosis in TGFb signaling has been extensively investigated [25], whereas endocytic regulation of the BMP signaling pathway is less studied. We have previously shown that Smad dependent signaling is initiated via PFCs at the plasma membrane and specific transcriptional activation is mediated through clathrin-dependent endocytosis [11]. In this study, we show that dynamin-dependent endocytosis is a prerequisite for a functional BMP-2 induced transcriptional cascade to induce the majority of BMP/Smad target genes. We demonstrate that expression of those genes is differentially affected by inhibition of endocytosis, leading to attenuation of osteoblast differentiation of C2C12 cells. We took advantage of the dynamin-dependent endocytosis inhibitor dynasore, which specifically interferes with the GTPase activity of dynamin and thereby inhibits pinching off of vesicles from the membrane, inhibiting both clathrin- and caveolae- mediated endocytosis [12]. Previous results from our group already demonstrated that Smad1/5/8 phosphorylation is induced at the plasma membrane as it was not affected by different endocytosis inhibitors when a single time point of 30 min BMP-2 stimulation was analyzed. Progression of the Smad dependent pathway by analysis of the transcriptional activation of the BRE-Luc reportergene was shown to be dependent on clathrin-mediated endocytosis [11]. Here, we expanded these studies using time-dependent assays of Smad phosphorylation. These studies confirmed that phosphorylation of Smad1/5/8 is still induced by BMP-2 when dynamin-dependent endocytosis is inhibited. However, new results from these studies are that dynasore treatment delays the dynamic profile and reduces the levels of pSmad1/5/8 formation (Figure 1C). Moreover, we show that the nuclear translocation dynamics of phosphorylated Smad1/5/8 are disturbed; the process was delayed, and the initial peak after 10 min of BMP-2 stimulation was missing (Figure 2A–C). In line with this finding, we demonstrated a down-regulation of BMP-2 induced activation of BRE-Luc activity in the presence of dynasore (Figure 2D,E). In TGFb signaling it was reported that Smad2 phosphorylation is a prerequisite for its detaching from the receptor and subsequent transcriptional activity [26,27]. Our results suggest that efficient phosphorylation of Smad1/5/8 and downstream signaling is PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e25163