title: Spatial segregation of BMP/Smad signaling affects osteoblast differentiation in C2C12 cells.
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.
  
  
  RESULTS
  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
  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.
authors: Institute for Chemistry and Biochemistry, Freie Universitaet Berlin, Berlin, Germany.
authors: Eva
authors: E
authors: Heining
authors: Raghu
authors: R
authors: Bhushan
authors: Pia
authors: P
authors: Paarmann
authors: Yoav I
authors: YI
authors: Henis
authors: Petra
authors: P
authors: Knaus
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000235:genetics; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D053263:Gene Regulatory Networks / Q000187:drug effects; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D005810:Multigene Family / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D019485:Bone Morphogenetic Proteins; D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D051785:Smad Proteins; D034281:Dynamins