Spatial Segregation of BMP/Smad Signaling Affects Osteoblast Differentiation in C2C12 Cells Eva Heining 1 , Raghu Bhushan 1 , Pia Paarmann 1 , Yoav I. Henis2, Petra Knaus1* 1 Institute for Chemistry and Biochemistry, Freie Universitaet Berlin, Berlin, Germany, 2 Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Abstract Background: Bone morphogenetic proteins (BMPs) are involved in a plethora of cellular processes in embryonic development and adult tissue homeostasis. Signaling specificity is achieved by dynamic processes involving BMP receptor oligomerization and endocytosis. This allows for spatiotemporal control of Smad dependent and non-Smad pathways. In this study, we investigate the spatiotemporal regulation within the BMP-induced Smad transcriptional pathway. Methodology/Principal Findings: Here we discriminate between Smad signaling events that are dynamin-dependent (i.e., require an intact endocytic pathway) and dynamin-independent. Inhibition of dynamin-dependent endocytosis in fluorescence microscopy and fractionation studies revealed a delay in Smad1/5/8 phosphorylation and nuclear translocation after BMP-2 stimulation of C2C12 cells. Using whole genome microarray and qPCR analysis, we identified two classes of BMP-2 induced genes that are differentially affected by inhibition of endocytosis. Thus, BMP-2 induced gene expression of Id1, Id3, Dlx2 and Hey1 is endocytosis-dependent, whereas BMP-2 induced expression of Id2, Dlx3, Zbtb2 and Krt16 is endocytosis-independent. Furthermore, we demonstrate that short term inhibition of endocytosis interferes with osteoblast differentiation as measured by alkaline phosphatase (ALP) production and qPCR analysis of osteoblast marker gene expression. Conclusions/Significance: Our study demonstrates that dynamin-dependent endocytosis is crucial for the concise spatial activation of the BMP-2 induced signaling cascade. Inhibition of endocytic processes during BMP-2 stimulation leads to altered Smad1/5/8 signaling kinetics and results in differential target gene expression. We show that interfering with the BMP-2 induced transcriptional network by endocytosis inhibition results in an attenuation of osteoblast differentiation. This implies that selective sensitivity of gene expression to endocytosis provides an additional mechanism for the cell to respond to BMP in a context specific manner. Moreover, we suggest a novel Smad dependent signal cascade induced by BMP-2, which does not require endocytosis. Citation: Heining E, Bhushan R, Paarmann P, Henis YI, Knaus P (2011) Spatial Segregation of BMP/Smad Signaling Affects Osteoblast Differentiation in C2C12 Cells. PLoS ONE 6(10): e25163. doi:10.1371/journal.pone.0025163 Editor: Neil A. Hotchin, University of Birmingham, United Kingdom Received May 25, 2011; Accepted August 26, 2011; Published October 5, 2011 Copyright: 2011 Heining et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the German-Israeli Foundation for Scientific Research and Development (GIF, grant number 932-244.13/2006 to PK and YIH). EH is a member of the Berlin-Brandenburg School for Regenerative Therapies (GSC 203). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: knaus@chemie.fu-berlin.de Introduction Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-b (TGF-b) superfamily and elicit important roles in proliferation, embryonic development, differ- entiation and tissue regeneration [1,2]. Urist and co-workers first identified BMPs as potent inducers of ectopic bone formation [3,4]. Accordingly, these cytokines were shown to posses the potential to convert mesenchymal cells into osteoblasts or chondroblasts [5]. BMPs signal via two transmembrane serine/threonine kinase receptors, the BMP receptors type I (BMPRIa, BMPRIb, Alk2) and the BMP receptor type II (BMPRII, ActRII and ActRIIB). BMP receptors form homomeric and heteromeric complexes that exist in distinct membrane areas and are differently modulated by their ligands. BMP-2 binds to preformed heterocomplexes (PFCs) of BMPRI and BMPRII, initiating Smad-dependent signaling. In contrast, ligand binding to the high affinity receptor BMPRI induces the formation of heteromeric BMP-induced signaling complexes (BISCs), which regulate non-Smad signaling. Signaling via BISCs activates the mitogen-activated protein kinase (MAPK) signaling cascade, leading to induction of alkaline phosphatase (ALP) expression [6]. The Smad pathway is initiated by phosphorylation of regulatory Smads (Smad1/5/8), which subse- quently associate with the common mediator Smad (Smad4), translocate into the nucleus, and regulate transcription of specific BMP target genes by recruiting additional activators or repressors [7]. Diseases ranging from skeletal diseases, vascular diseases, tissue dystrophies to cancer are caused by malfunctions of BMP signaling pathways. This highlights the importance of fine-tuning the BMP signaling responses. Extracellular antagonists such as Noggin control binding of the ligand to the receptor complexes [8]. Co- receptors like Ror2 and DRAGON or cytoplasmic co-regulators PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e25163