Spatial segregation of BMP/Smad signaling affects osteoblast differentiation in C2C12 cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE29373 | GEO | 2012/02/16
SECONDARY ACCESSION(S): PRJNA140215
REPOSITORIES: GEO
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Project description: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.
| S-EPMC3187766 | biostudies-literature
Project description:Analysis of BMP-2 induced gene expression of C2C12 cells treated with the endocytosis inhibitor dynasore to identify gene expression profiles which are sensitive to endocytosis inhibition. Our data highlight the importance of endocytosis for transmission of an extracellular BMP-2 signal into the nucleus to activate a transcriptional gene network Total RNA obtained from C2C12 cells stimulated with BMP-2 together with the endocytosis inhibitor dynasore or DMSO as control compared to unstimulated cells treated with dynasore or DMSO alone.
2012-02-16 | E-GEOD-29373 | biostudies-arrayexpress
Project description:Gene-specific transcription factors (GSTFs) control of gene transcription by DNA binding and specific protein complex recruitment, which regulates promoter accessibility for transcription initiation by RNA polymerase II. GSTFs that are frequently mutated in colon and rectal carcinomas are Suppressor of Mothers Against Decapentaplegic 2 (SMAD2) and SMAD4, which play an important role in the TGF-β signaling pathways controlling cell fate and proliferation (ref.). The SMAD protein family is a diverse and it can be divided into three subclasses: receptor activated SMADs, inhibitory SMADs and the common SMAD4 co-activator. To study protein interactors of the SMAD protein family we generated a quantitative proteomics pipeline that allows for inducible expression of GFP-tagged SMAD proteins followed by affinity purification and MS analysis. The nuclear importin IPO5 was identified as a novel interacting protein of SMAD1. Overexpression of IPO5 shows forced BMP R-SMAD nuclear localization confirming a functional relationship between BMP but not TGF-β R-SMADs and IPO5. Finally we provide evidence that the length of the lysine stretch in the NLS is involved in importin selection.
2016-10-10 | PXD004529 | Pride
Project description:Bone morphogenetic proteins (BMPs) are key regulators of cell fate decisions during embryogenesis and tissue homeostasis. BMPs signal through a coordinated assembly of two types of transmembrane serine/threonine kinase receptors to induce Smad1/5/8 plus non-Smad pathways, such as MAPK and Akt. The recent discovery of BMP receptor inhibitors opened new avenues to study specific BMP signalling and to delineate this effect from TGF-? and Activin signalling. Here we present comprehensive and quantitative analyses on both canonical and non-Smad mediated BMP signalling under Dorsomorphin (DM) and LDN-193189 (LDN) treatment conditions. We demonstrate for the first time, that both compounds affect not only the Smad but also the non-Smad signalling pathways induced by either BMP2, BMP6 or GDF5. The activation of p38, ERK1/2 and Akt in C2C12 cells was inhibited by DM and LDN. In addition "off-target" effects on all branches of BMP non-Smad signalling are presented. From this we conclude that the inhibition of BMP receptors by DM and more efficiently by LDN-193189 affects all known BMP induced signalling cascades.
| S-EPMC6164168 | biostudies-literature
Project description:Bone morphogenetic proteins (BMPs) regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. We used cDNA microarrays to elucidate regulators of BMP-2-induced osteoblast differentiation.
2011-11-11 | E-GEOD-33567 | biostudies-arrayexpress
Project description:The aim of this study was to investigate if chemically produced nanotopography on titanium (Ti) surface induces osteoblast differentiation of cultured human bone marrow mesenchymal stem cells (hMSCs) by regulating the expression of microRNAs (miRs). It was demonstrated that Ti with nanotopography induces osteoblast differentiation of hMSCs as evidenced by upregulation of osteoblast specific markers compared with untreated (control) Ti at day 4. At this time-point, miR-sequencing analysis revealed that 20 miRs were upregulated (>twofold) while 20 miRs were downregulated (>threefold) in hMSCs grown on Ti with nanotopography compared with control Ti. Three miRs, namely miR-4448, -4708, and -4773, which were significantly downregulated (>fivefold) by Ti with nanotopography affect osteoblast differentiation of hMSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP-2) osteogenic signal, which were upregulated by Ti with nanotopography. Overexpression of miR-4448, -4708, and 4773 in MC3T3-E1 pre-osteoblasts noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and therefore repressed the gene expression of key bone markers. Additionally, it was observed that the treatment with BMP-2 displayed a higher osteogenic effect on MC3T3-E1 cells grown on Ti with nanotopography compared with control Ti, suggesting that the BMP-2 signaling pathway was more effective on this surface. Taken together, these results indicate that a complex regulatory network involving a miR-SMAD-BMP-2 circuit governs the osteoblast differentiation induced by Ti with nanotopography. J. Cell. Physiol. 229: 1690-1696, 2014. © 2014 Wiley Periodicals, Inc.
| S-EPMC4926039 | biostudies-literature
Project description:Here we describe three new small-molecule activators of BMP signaling found by high throughput screening of a library of ~600,000 small molecules. Using a cell-based luciferase assay in the BMP4-responsive human cervical carcinoma clonal cell line, C33A-2D2, we identified three compounds with similar chemotypes, each ventralized zebrafish embryos and stimulated increased expression of the Bmp target genes, bmp2b and szl. Because these compounds ventralize zebrafish embryos, we have termed them “ventromorphins.” As expected for a BMP pathway activator, they induce the differentiation of C2C12 myoblasts to osteoblasts. Affymetrix RNA analysis confirmed the differentiation results and showed that ventromorphin treatments elicits a genetic response similar to BMP-4 treatment. Unlike isoliquiritigenin (SJ000286273), a flavone that maximally activates the pathway after 24 hours of treatment, all three ventromorphins induced SMAD1/5/8 phosphorylation within 30 minutes of treatment and achieved peak activity at 30 minutes or 1 hour, indicating that their direct responses are consistent with activated BMP signaling.
2018-02-14 | GSE95248 | GEO
Project description:Mindbomb1 is a Novel Negative Regulator of BMP Signaling.
2024-07-02 | MSV000095215 | MassIVE
Project description:Vascular calcification is an important pathological condition associated with increased risk of cardiovascular mortality. Hydroxyapatite (HA) found in such deposits is the same polymorph of calcium (Ca) found in bone, indicating calcification may involve mechanisms akin to bone formation. Vascular smooth muscle cells (Vsmcs) have been shown to undergo phenotypic change to osteoblast-like cells. However, the mechanisms underlying this phenotypic change are unclear, and whether the stimulus to become osteogenic is a result of loss of mineralization inhibitors or early mineral deposits is not known. Our aim in this study is to identify mechanisms and signal transduction pathways that cause differentiation of Vsmcs into osteoblast-like cells in the presence of HA. We first characterized vascular origin of Vsmcs by studying the expression of smooth muscle cell markers: myosin heavy chain and smooth muscle actin along with SM22? at both mRNA and protein levels. Vsmcs grown on HA exhibited progressive change in cellular morphology at 3-, 7-, and 14-day time points. Culturing of Vsmcs on HA disc resulted in decrease in media Ca levels and increased expression of Ca-sensing receptor (CaSR) on Vsmcs resulting in upregulation of intracellular CaSR signaling leading to increased BMP-2 secretion. BMP-2 pathway mediated differentiation of Vsmcs to osteoblast-like cells shown by expression of osteogenic markers like runt-related transcription factor 2, osteocalcin, and alkaline phosphatase at mRNA and protein levels. Blocking CaSR by NPS-2143 reduced BMP-2 secretion and blocking the BMP-2 pathway by LDN-193189, a BMP inhibitor, modulated expression of osteogenic markers confirming their role in osteogenesis of Vsmcs.
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Project description:Fracture healing is related to osteogenic differentiation and mineralization. Recently, due to the unwanted side effects and clinical limitations of existing treatments, various natural product-based chemical studies have been actively conducted. Albiflorin is a major ingredient in Paeonia lactiflora, and this study investigated its ability to promote osteogenic differentiation and fracture healing. To demonstrate the effects of albiflorin on osteoblast differentiation and calcified nodules, alizarin red S staining and von Kossa staining were used in MC3T3-E1 cells. In addition, BMP-2/Smad and Wnt/β-catenin mechanisms known as osteoblast differentiation mechanisms were analyzed through RT-PCR and western blot. To investigate the effects of albiflorin on fracture healing, fractures were induced using a chainsaw in the femur of Sprague Dawley rats, and then albiflorin was intraperitoneally administered. After 1, 2, and 3 weeks, bone microstructure was analyzed using micro-CT. In addition, histological analysis was performed by staining the fractured tissue, and the expression of osteogenic markers in serum was measured. The results demonstrated that albiflorin promoted osteoblastogenesis and the expression of RUNX2 by activating BMP-2/Smad and Wnt/β-catenin signaling in MC3T3-E1 cells. In addition, albiflorin upregulated the expression of various osteogenic genes, such as alkaline phosphatase, OCN, bone sialoprotein, OPN, and OSN. In the femur fracture model, micro-CT analysis showed that albiflorin played a positive role in the formation of callus in the early stage of fracture recovery, and histological examination proved to induce the expression of osteogenic genes in femur tissue. In addition, the expression of bone-related genes in serum was also increased. This suggests that albiflorin promotes osteogenesis, bone calcification and bone formation, thereby promoting the healing of fractures in rats.
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