Elsevier

Bone

Volume 50, Issue 5, May 2012, Pages 1092-1099
Bone

Original Full Length Article
Ubc9 negatively regulates BMP-mediated osteoblastic differentiation in cultured cells

https://doi.org/10.1016/j.bone.2012.02.008Get rights and content

Abstract

SUMO (small ubiquitin-related modifier) modification (SUMOylation) has been reported to regulate various biological events such as cell-cycle progression, proliferation, and survival. Bone morphogenetic proteins (BMPs) play an important role in osteoblast differentiation and maturation. Although Smad4, which acts as a transcriptional factor in the BMP signaling, is a target of SUMOylation, the involvement of SUMOylation in osteoblast differentiation remains unclear. In this report, we demonstrated spatial expression patterns of SUMO proteins and Ubc9 (ubiquitin conjugating enzyme 9), which is a unique E2-SUMOylation enzyme, in mouse tibia. Furthermore, siRNA knockdown of Ubc9 enhanced osteoblastic differentiation induced by BMP2 in C2C12 mouse myoblasts and ST2 mouse bone-marrow derived stromal cells. Ubc9 knockdown elevated the BMP signaling transduction and reduced the expression of muscle-related genes in cooperation with BMP2. Finally, a luciferase assay using an Id1 (target gene of BMP signaling) reporter revealed that Smad4 mutants prevented from SUMOylation at their Lys158 possessed more potent transcriptional activity than wild-type Smad4. Taken together, these findings suggest that Ubc9 negatively regulates osteoblastic differentiation induced by BMP via, at least in part, SUMOylation of Smad4.

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.

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