Young-Dan Cho

Molecular Regulation of Matrix Extracellular Phosphoglycoprotein Expression by Bone Morphogenetic Protein-2

Matrix extracellular phosphoglycoprotein (MEPE) is mainly expressed in mineralizing tissues, and its C-terminal proteolytic cleavage product is an acidic-serine-asparate-rich-MEPE-associated motif (ASARM) that is a strong regulator of body phosphate metabolism and mineralization. There is sufficient data supporting a role for MEPE protein function in mineralization, however, little is known about the regulation of MEPE gene expression. As bone morphogenetic protein-2 (BMP-2) is one of the most important signals for calvarial mineralization and MEPE expression is higher in mineralized tissues, we attempted to uncover a regulatory circuit between BMP-2 and MEPE expression. Mepe expression is very low in proliferating MC3T3-E1 cells, but is dramatically increased in the mineralization stage and is strongly stimulated by treatment with BMP-2, even in proliferating cells. Overexpression and knock-down experiments of Smads, Dlx5, and Runx2 indicated that they are indispensable mediators of BMP-2-induced Mepe expression. In contrast, Msx2 showed strong inhibition of Mepe transcription. PHEX is an enzyme that prevents the release of the ASARM motif, a mineralization inhibitor, from the MEPE molecule. Thus, the MEPE/PHEX ratio may be a good indicator of mineralization progression because we found that the mRNA ratio and protein levels were low when osteoblasts were actively differentiating to the mineralization stage and the ratio was high when the cells reached the mineralization stage when it is assumed that osteocytes may protect themselves and make a space to survive from the mineralized matrix by releasing the ASARM motif. Collectively, MEPE expression is bone cell-specific and induced by the BMP-2 signaling pathway. In addition, the MEPE/PHEX ratio of the cell could be a very important barometer indicating the progression of tissue mineralization.

The Canonical BMP Signaling Pathway Plays a Crucial Part in Stimulation of Dentin Sialophosphoprotein Expression by BMP-2

Dentin sialophosphoprotein (DSPP), a typical dentin-specific protein, is mainly expressed in the dentin extracellular matrix and plays a role in dentin mineralization. BMP-2 provides a strong signal for differentiation and mineralization of odontoblasts and osteoblasts. Previously, BMP-2 treatment is reported to stimulate Dspp expression in the MD10-F2 pre-odontoblast cells through activation of the heterotrimeric transcription factor Y (NF-Y). The canonical BMP signaling pathway is known to contribute greatly to biomineralization, however, it is not known whether it is involved in Dspp expression. Here, we investigated this question. Activation of the canonical BMP-2 signaling pathway in MDPC-23, preodontoblast cell, by overexpression of constitutively active Smad1/5 or downstream transcription factors Dlx5 and Runx2 stimulated Dspp expression. Conversely, knockdown of each element with siRNA significantly blocked the BMP-2-induced Dspp expression. To test whether these transcription factors downstream of BMP-2 are directly involved in regulating Dspp, we analyzed the mouse Dspp promoter. There are 5 well conserved homeodomain binding elements, H1 to H5, in Dspp proximal promoter regions (−791 to +54). A serial deletion of H1 and H2 greatly changed basal promoter activity and responsiveness to Dlx5 or Msx2. However, further deletions did not change the responsiveness to Dlx5 or Msx2. H1 and H2 sites can be suggested as specific response elements of Dlx5 and Msx2, respectively, based on their promoter activity modulation. Thus, the canonical BMP-2 signaling pathway plays a crucial part in the regulation of Dspp expression through the action of Smads, Dlx5, Runx2, and Msx2.

The Boston-type Craniosynostosis Mutation MSX2 (P148H) Results in Enhanced Susceptibility of MSX2 to Ubiquitin-dependent Degradation

Boston-type craniosynostosis is caused by a single amino acid substitution, P148H, in the transcription factor MSX2. The increased binding affinity of MSX2 (P148H) to the response element has led many to hypothesize that the substitution is a gain-of-function mutation. However, there have been conflicting reports on the function of MSX2, and by extension, the nature of the P148H mutation remains unclear. In this study, we have examined the molecular mechanism of MSX2 function and the nature of the P148H mutation. During cranial suture closure of rodent, Msx2 expression was detected in the suture space. Overexpression of wild type MSX2 in mesenchymal cells stimulated cell proliferation and cyclin D1 expression, whereas P148H mutant did not. These results indicated that MSX2 is involved in maintaining the suture space by stimulating suture mesenchymal cell proliferation and that P148H is defective in this process. The protein levels of P148H were lower than wild type Msx2 (Msx2-WT), and pulse-chase experiments indicated that the mutant protein has a shorter half-life than the Msx2-WT protein. The ubiquitylation level of P148H was greater than that of Msx2-WT. The degradation of Msx2 was mediated by Praja1, and the P148H mutant was degraded more effectively than WT. The ubiquitylation of Msx2-WT was higher in the presence of Msx2 (P148H), which indicated that P148H functions as a dominant-negative mutant. Collectively, the primary function of MSX2 in suture closure is the induction of cell proliferation and suture maintenance, and the mutation results in an increased susceptibility of both wild type and mutant MSX2 to proteasomal degradation.

Prolyl isomerase Pin1-mediated conformational change and subnuclear focal accumulation of Runx2 are crucial for fibroblast growth factor 2 (FGF2)-induced osteoblast differentiation.

Background: Genetic interaction between Runx2 and Pin1 is critical for embryonic bone formation. Results: Pin1 is a critical modifying enzyme promoting both subnuclear accumulation and protein acetylation of Runx2. Conclusion: Pin1 determines the fate of Runx2 protein in osteoblast differentiation. Significance: The modulation of Pin1 activity may be a clinical target for the regulation of bone formation. Fibroblast growth factor 2 (FGF2) signaling plays a pivotal role in bone growth/differentiation through the activation of osteogenic master transcription factor Runx2, which is mediated by the ERK/MAPK-dependent phosphorylation and the p300-dependent acetylation of Runx2. In this study, we found that Pin1-dependent isomerization of Runx2 is the critical step for FGF2-induced Runx2 transactivation function. We identified four serine or threonine residues in the C-terminal domain of Runx2 that are responsible for Pin1 binding and structural modification. Confocal imaging studies indicated that FGF2 treatment strongly stimulated the focal accumulation of Pin1 in the subnuclear area, which recruited Runx2. In addition, active forms of RNA polymerase-II also colocalized in the same subnuclear compartment. Dipentamethylene thiuram monosulfide, a Pin1 inhibitor, strongly attenuated their focal accumulation as well as Runx2 transactivation activity. The Pin1-mediated structural modification of Runx2 is an indispensable step connecting phosphorylation and acetylation and, consequently, transcriptional activation of Runx2 by FGF signaling. Thus, the modulation of Pin1 activity may be a target for the regulation of bone formation.

Wnt3a stimulates Mepe, Matrix extracellular phosphoglycoprotein, expression directly by the activation of the canonical Wnt signaling pathway and indirectly through the stimulation of autocrine Bmp-2 expression

Matrix extracellular phosphoglycoprotein (MEPE) is a specific marker of mineralizing osteoblasts and osteocytes. Canonical BMP and Wnt signaling pathways are two of the strongest paracrine signals stimulating osteogenesis. Our previous results indicated that Mepe expression is stimulated by the BMP‐2‐signaling pathway. The specific aim of this study addressed whether Mepe expression is also controlled by Wnt signaling, and whether there is a cross‐regulation between two major osteogenic signaling pathways. Treatment with Wnt3a, a canonical Wnt signaling stimulator, strongly enhanced Mepe mRNA expression. Knock‐down of β‐catenin with siRNA completely reversed Wnt3a‐stimulated Mepe expression. The Mepe mRNA expression level was increased by overexpression of β‐catenin and Lef‐1, even in the absence of Wnt3a. Highly conserved Lef‐1 response elements were identified in the mouse Mepe promoter. The direct binding of Lef‐1 to these elements is critical for Mepe expression, indicating that Mepe is a direct target of canonical Wnt signaling. Meanwhile, we also found that Wnt3a treatment strongly stimulated Bmp‐2 expression, and that the subsequent increase in Bmp‐2 protein was determined in Wnt3a‐treated conditioned medium (CM). Treatment of MC3T3‐E1 cells with CM stimulated phosphorylation of the Smad1/5 proteins and their downstream Dlx5 mRNA expression. The CM‐mediated increases of phospho‐Smad and Dlx5 expression were not blocked completely by a Wnt3a antagonist, Dkk‐1, but were almost completely suppressed by the addition of a Bmp‐2 antagonist, Noggin. Collectively, Wnt3a stimulates Mepe transcription directly by a canonical Wnt signaling pathway through β‐catenin and Lef‐1 and indirectly through the activation of a Bmp‐2 autocrine loop. J. Cell. Physiol. 227: 2287–2296, 2012.

Pin1-mediated Modification Prolongs the Nuclear Retention of β-Catenin in Wnt3a-induced Osteoblast Differentiation.

The canonical Wnt signaling pathway, in which β-catenin nuclear localization is a crucial step, plays an important role in osteoblast differentiation. Pin1, a prolyl isomerase, is also known as a key enzyme in osteogenesis. However, the role of Pin1 in canonical Wnt signal-induced osteoblast differentiation is poorly understood. We found that Pin1 deficiency caused osteopenia and reduction of β-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear β-catenin level, TOP flash activity, and expression of bone marker genes induced by canonical Wnt activation and vice versa in Pin1 overexpression. Pin1 interacts directly with and isomerizes β-catenin in the nucleus. The isomerized β-catenin could not bind to nuclear adenomatous polyposis coli, which drives β-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of β-catenin in the nucleus and might explain the decrease of β-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate β-catenin-mediated osteogenesis.

Pin1 plays a critical role as a molecular switch in canonical BMP signaling.

Pin1 is a peptidyl prolyl cis‐trans isomerase that specifically binds to the phosphoserine–proline or phosphothreonine–proline motifs of numerous proteins. Previously, we reported that Pin1 deficiency resulted in defects in osteoblast differentiation during early bone development. In this study, we found that adult Pin1‐deficient mice developed osteoporotic phenotypes compared to age‐matched controls. Since BMP2 stored in the bone matrix plays a critical role in adult bone maintenance, we suspected that BMP R‐Smads (Smad1 and Smad5) could be critical targets for Pin1 action. Pin1 specifically binds to the phosphorylated linker region of Smad1, which leads to structural modification and stabilization of the Smad1 protein. In this process, Pin1‐mediated conformational modification of Smad1 directly suppresses the Smurf1 interaction with Smad1, thereby promoting sustained activation of the Smad1 molecule. Our data demonstrate that post‐phosphorylational prolyl isomerization of Smad1 is a converging signal to stabilize the Smad1 molecule against the ubiquitination process mediated by Smurf1. Therefore, Pin1 is a critical molecular switch in the determination of Smad1 fate, opposing the death signal transmitted to the Smad1 linker region by phosphorylation cascades after its nuclear localization and transcriptional activation. Thus, Pin1 could be developed as a major therapeutic target in many skeletal diseases. J. Cell. Physiol. 230: 640–647, 2015.

Epigenetic Priming Confers Direct Cell Trans-Differentiation From Adipocyte to Osteoblast in a Transgene-Free State

The bone marrow of healthy individuals is primarily composed of osteoblasts and hematopoietic cells, while that of osteoporosis patients has a larger portion of adipocytes. There is evidence that the epigenetic landscape can strongly influence cell differentiation. We have shown that it is possible to direct the trans‐differentiation of adipocytes to osteoblasts by modifying the epigenetic landscape with a DNA methyltransferase inhibitor (DNMTi), 5′‐aza‐dC, followed by Wnt3a treatment to signal osteogenesis. Treating 3T3‐L1 adipocytes with 5′‐aza‐dC induced demethylation in the hypermethylated CpG regions of bone marker genes; subsequent Wnt3a treatment drove the cells to osteogenic differentiation. When old mice with predominantly adipose marrow were treated with both 5′‐aza‐dC and Wnt3a, decreased fatty tissue and increased bone volume were observed. Together, our results indicate that epigenetic modification permits direct programming of adipocytes into osteoblasts in a mouse model of osteoporosis, suggesting that this approach could be useful in bone tissue‐engineering applications. J. Cell. Physiol. 231: 1484–1494, 2016.

An HDAC Inhibitor, Entinostat/MS-275, Partially Prevents Delayed Cranial Suture Closure in Heterozygous Runx2 Null Mice

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal disorder caused by mutations in RUNX2, coding a key transcription factor of early osteogenesis. CCD patients suffer from developmental defects in cranial bones. Despite numerous investigations and clinical approaches, no therapeutic strategy has been suggested to prevent CCD. Here, we show that fetal administration of Entinostat/MS‐275, a class I histone deacetylase (HDAC)‐specific inhibitor, partially prevents delayed closure of cranial sutures in Runx2+/‐ mice strain of C57BL/6J by two mechanisms: 1) posttranslational acetylation of Runx2 protein, which stabilized the protein and activated its transcriptional activity; and 2) epigenetic regulation of Runx2 and other bone marker genes. Moreover, we show that MS‐275 stimulates osteoblast proliferation effectively both in vivo and in vitro, suggesting that delayed skeletal development in CCD is closely related to the decreased number of progenitor cells as well as the delayed osteogenic differentiation. These findings provide the potential benefits of the therapeutic strategy using MS‐275 to prevent CCD.

An Overview of Biomaterials in Periodontology and Implant Dentistry

Material is a crucial factor for the restoration of the tooth or periodontal structure in dentistry. Various biomaterials have been developed and clinically applied for improved periodontal tissue regeneration and osseointegration, especially in periodontology and dental implantology. Furthermore, the biomimetic approach has been the subject of active research in recent years. In this review, the most widely studied biomaterials (bone graft material, barrier membrane, and growth or differentiation factors) and biomimetic approaches to obtain optimal tissue regeneration by making the environment almost similar to that of the extracellular matrix are discussed and specifically highlighted.