Spatial Segregation of BMP/Smad Signaling Affects Osteoblast Differentiation in C2C12 Cells

Abstract

BackgroundBone 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 FindingsHere 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/SignificanceOur 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.

BMP2 Increases Adipogenic Differentiation in the Presence of Dexamethasone, which is Inhibited by the Treatment of TNF-a in Human Adipose Tissue-Derived Stromal Cells

“…Zuk et al reported a defect of BMP2 signaling in hADSCs [13]. However, we observed expression of various isoforms of BMP receptors and increase in expression of BMP2 downstream target genes such as DLX3 and ID2 [23] as like in hBMSCs. BMPs binding induces formation of heteromultimers of both type I and II receptors [24].…”

Fish bone peptide promotes osteogenic differentiation of MC3T3‐E1 pre‐osteoblasts through upregulation of MAPKs and Smad pathways activated BMP‐2 receptor

“…The activation of MAPK signalling cascade contributes to the induction of expression of the phenotypic markers of osteoblastic differentiation . Additionally, the Smad pathway is also affected during osteoblast differentiation through phosphorylation of regulatory Smads . We had shown that FBP‐KSA induced phosphorylation of MAPKs (p38, ERK, and JNK) and Smads.…”

Osteoblast differentiation and migration are regulated by dynamin GTPase activity

“…However, it appears that dynamin activity is dispensable for latter stages of osteoblast differentiation as indicated by the finding that long-term incubation with dynasore (3 days) had little effect on ALP expression, and collagen and osteocalcin mRNA levels were unaffected by dynasore. In studies published by others (Heining et al, 2011), it was reported that 40 μM dynasore blocked the ability of the bone morphogenic protein 2 (BMP-2) to induce transcription in the osteoblastic cell line, C2C12, which was suggested to attenuate osteoblast differentiation. However, no dose-response analyses were performed, and we found a clear temporal and dose-dependent effect of dynasore on osteoblast differentiation.…”