Original Full Length ArticleUbc9 negatively regulates BMP-mediated osteoblastic differentiation in cultured cells
Highlights
► siRNA knockdown of Ubc9 enhanced BMP2-induced osteoblastic differentiation in C2C12 cells. ► Reporter assay showed that Ubc9 knockdown directly enhances the BMP signal transduction. ► Inhibition of SUMOylation of Smad4 elevated its transcriptional activity. ► Our data suggest that SUMOylation negatively regulated BMP-induced osteoblastic differentiation.
Introduction
The bone morphogenetic proteins (BMPs) proteins were originally identified as proteins that induce ectopic bone formation in vivo [1], [2]. BMPs are members of the transforming growth factor-β (TGF-β) super family, which plays a crucial role in many biological events such as differentiation, survival, and proliferation. Secreted BMPs bind to BMP type I and type II receptors, which are serine/threonine kinases. By this binding, the BMP type I receptor is phosphorylated by the type II receptor, and then the activated type I receptor phosphorylates receptor regulated Smads (R-Smads; Smad1/5/8). Phosphorylated R-Smads form complexes with common Smad (Co-Smad; Smad4 Refs. [3], [4]). These complexes are translocated into the nucleus where they regulate the transcription of various target genes, including Id1, which encodes a myogenic differentiation inhibitor [5]. BMP signaling also induces the expression of runt-related transcription factor 2 (Runx2, also called Cbfa1) and Osterix (Sp7), which are key transcription factors for osteoblastic differentiation and bone mineralization [6].
Smad4 acts as a transcriptional factor in both BMP and TGF-β signaling and is one of the target proteins of SUMO (small ubiquitin-related modifier) conjugation. The SUMO protein has homology to ubiquitin. This protein is present in all eukaryotes and highly conserved from yeast to humans. Three SUMO proteins (SUMO-1, SUMO-2, SUMO-3) have been identified in vertebrates, and one in invertebrates [7], [8]. SUMO conjugation (SUMOylation) is an ubiquitination-like post-translational modification associated with many cellular functions. Although SUMOylation resembles ubiquitination, enzymes different from those participating in ubiquitination are involved in SUMOylation. The AOS/UBA2 heterodimer adenylates and activates the SUMO molecule, similar to the action of E1-ubiqutination enzymes [9], [10]. Ubiquitin conjugating enzyme 9 (Ubc9) functions as the only E2-SUMOylation enzyme (conjugating enzyme; Refs. [11], [12], [13], [14]).
To date, many proteins including transcriptional factors such as Smad4 have been reported to be SUMOylated. Previous studies reported that SUMOylation of Smad4 negatively [15], [16] or positively [17], [18], [19] regulates its transcriptional activity induced by TGF-β. Recently, the role of SUMOylation in BMP signaling was reported. Shimada and his colleagues reported that knockdown of Ubc9 in human osteoblast-like Saos-2 cells inhibits the expression of BMP-induced genes and reduces protein level of Smad4 and phosphorylation of Smad1 [20]. This report suggests that Ubc9 is necessary for BMP signaling transduction, although another group reported that Ubc9 negatively regulates BMP signaling in Drosophila development. That study showed that the expression pattern of Decapentaplegic (Dpp, a BMP signaling molecule in Drosophila) target genes is expanded in embryos with a Ubc9 mutation and that SUMOylation of Medea (Drosophila ortholog of Smad4) negatively regulates the Dpp signaling by controlling the subcellular distribution of Medea [21]. Although these studies suggest that SUMOylation plays a role in the BMP signaling pathway, it remains unclear whether SUMOylation affects on osteoblastic differentiation. To this end, we investigated whether inhibition of SUMOylation by knockdown of Ubc9 would affect BMP-induced osteoblastic differentiation in C2C12 mouse myoblast cells.
Section snippets
Animals
Two-month-old male C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). All procedures for animal care were approved by the Animal Management Committee of Matsumoto Dental University.
Immunohistochemistry
Mouse tibias were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.05 M phosphate buffer (pH 7.4) and then demineralized with 10% EDTA for 3 weeks. After dehydration by soaking in graded ethanol, the samples were embedded in paraffin and sliced with a microtome. The sections were pretreated with
Localization of proteins involved in SUMOylation in bone cells
To evaluate the contribution of SUMOylation to bone formation, we investigated the localization of 3 subtypes of SUMO proteins (SUMO-1, -2, -3) and the unique SUMO conjugating enzyme (Ubc9) in mouse tibia by performing immunohistochemistry. Immunoreactivities for SUMOs and Ubc9 were detected in the nucleus of osteocytes and of some fibroblastic cells in contact with osteoblasts, whereas the label was scarcely seen in osteoblasts on the bone surface (Fig. 1). No obvious differences were observed
Discussion
Our immunohistochemical data showed that SUMO proteins and Ubc9 are localized weakly in osteoblasts whereas clear localization of these proteins is observed in fibroblastic cells residing in the bone marrow, which population is thought to include osteoblast precursor cells (Fig. 1). From these findings, we predicted that SUMOylation may negatively regulate the early differentiation of osteoblasts. To clarify the role of SUMOylation in the early differentiation of osteoblasts, we tested Ubc9
Acknowledgments
We thank Drs. Tadashi Ninomiya, Yasuhiro Kobayashi, Kazuhiro Maeda, Yasuhiro Imamura, Yosuke Funato, Satoshi Ohte, Tatsuo Michiue, Hiroki Danno, and members of the Institute for Oral Science, Masumoto Dental University for their valuable advice, comments, and discussion. We are grateful to Drs. Akira Kikuchi and Peter ten Dijke for providing the constructs used. This work was supported in part by grant-in-aids from the Ministry of Education, Culture, Sports, Science, and Technology.
References (35)
- et al.
Bone morphogenetic proteins: a critical review
Cell Signal
(2011) - et al.
BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk
Cytokine Growth Factor Rev
(2005) - et al.
Ubiquitin-like proteins: new wines in new bottles
Gene
(2000) - et al.
Molecular cloning and characterization of human AOS1 and UBA2, components of the sentrin-activating enzyme complex
FEBS Lett
(1999) - et al.
Ubch9 conjugates SUMO but not ubiquitin
FEBS Lett
(1997) - et al.
Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9
J Biol Chem
(1997) - et al.
Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p
J Biol Chem
(1997) - et al.
Daxx mediates the small ubiquitin-like modifier-dependent transcriptional repression of Smad4
J Biol Chem
(2005) - et al.
Activation of transforming growth factor-beta signaling by SUMO-1 modification of tumor suppressor Smad4/DPC4
J Biol Chem
(2003) - et al.
Transforming growth factor-beta-mediated signaling via the p38 MAP kinase pathway activates Smad-dependent transcription through SUMO-1 modification of Smad4
J Biol Chem
(2003)
Regulation of Smad4 sumoylation and transforming growth factor-beta signaling by protein inhibitor of activated STAT1
J Biol Chem
Ubc9 promotes the stability of Smad4 and the nuclear accumulation of Smad1 in osteoblast-like Saos-2 cells
Bone
Sumoylation of Smad4, the common Smad mediator of transforming growth factor-beta family signaling
J Biol Chem
The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice
Dev Cell
Ubc9 expression is essential for myotube formation in C2C12
Exp Cell Res
SUMO promotes HDAC-mediated transcriptional repression
Mol Cell
Bone: formation by autoinduction
Science
Cited by (13)
Regulation of BMP-induced osteoblastic differentiation by Ubc9 knockdown-mediated inhibition of SUMO modification
2014, Journal of Oral BiosciencesCitation Excerpt :In contrast, this protein is scarcely detected in osteoblasts on the bone surface. The localization of other subtypes of SUMO (SUMO-2/3) and the unique SUMO-conjugation enzyme (Ubc9) in bone resemble that of SUMO-1 [38]. These findings suggest that the SUMOylation pathway is inhibited in osteoblasts.
Roles of Vascular Smooth Muscle Cells in Atherosclerotic Calcification
2023, Journal of Lipid and AtherosclerosisChemokine ligand 28 (CCL28) negatively regulates trabecular bone mass by suppressing osteoblast and osteoclast activities
2021, Journal of Bone and Mineral Metabolism