Hyuck Choi

Sustained BMP-2 delivery and injectable bone regeneration using thermosensitive polymeric nanoparticle hydrogel bearing dual interactions with BMP-2.

Localized and continuous osteogenic stimulation to defected sites is required for effective bone regeneration. Here, we suggest an injectable and sustained bone morphogenetic protein-2 (BMP-2) release system using thermosensitive polymeric nanoparticles bearing dual interacting forces with BMP-2. For sustained BMP-2 release, hydrophobic and ionic interactions were introduced to thermosensitive poly(phosphazene). Hydrophobic isoleucine ethyl ester and hydrophilic poly-ethylene glycol were mainly substituted to the poly(phosphazene) back bone for amphiphilicity and hydrophobic interaction with BMP-2. Carboxylic acid moiety was additionally substituted to the back bone for ionic interaction with BMP-2. These dual interacting polymeric nanoparticles (D-NPs) formed compact nanocomplexes with BMP-2. The aqueous solution of BMP-2/D-NP nanocomplexes was transformed to hydrogel when the temperature of the solution increased. Loaded BMP-2 was sustain-released for three weeks from the BMP-2/D-NP nanocomplex hydrogel. The extended BMP-2 exposure caused higher osteocalcin secretion in C2C12 cells. Significant bone generations were observed at the target site by single injection of BMP-2/D-NP nanocomplexes in vivo.

MicroRNA-302a Stimulates Osteoblastic Differentiation by Repressing COUP-TFII Expression

Chicken ovalbumin upstream promoter transcription factor II (COUP‐TFII) is a potent transcription factor that represses osteoblast differentiation and bone formation. Previously, we observed that stimuli for osteoblast differentiation, such as bone morphogenetic protein 2 (BMP2), inhibits COUP‐TFII expression. This study was undertaken to identify BMP2‐regulated and COUP‐TFII‐targeting microRNAs (miRNAs), and to explore their regulatory roles in osteoblast differentiation. Based on in silico analysis, 12 miRNAs were selected and their expression in BMP2‐treated MC3T3‐E1 cells was examined. BMP2 induced miR‐302a expression in dose‐ and time‐dependent manners with the decrease in COUP‐TFII expression. Runx2, a BMP2‐downstream transcription factor, specifically regulated miR‐302a expression and its promoter activity. A computer‐based prediction algorithm led to the identification of two miR‐302a binding sites on the 3′‐untranslational region of COUP‐TFII mRNA (S1: 620–626 bp, S2: 1,016–1,022 bp), and a luciferase assay showed that miR‐302a directly targeted S1 and S2. Transfection of miR‐302a precursor significantly enhanced expression of osteogenic marker genes with decreasing COUP‐TFII mRNA and protein level, alkaline phosphatase activity and matrix mineralization. On the other hand, inhibition of miR‐302a significantly attenuated BMP2‐induced osteoblast specific gene expression, alkaline phosphatase activity, and matrix mineralization with increasing COUP‐TFII mRNA and protein level. These results indicate that miR‐302a is induced by osteogenic stimuli and promotes osteoblast differentiation by targeting COUP‐TFII. MiR‐302a could be a positive regulator for osteoblast differentiation. J. Cell. Physiol. 230: 911–921, 2015.

Transcriptional Factor ATF6 is Involved in Odontoblastic Differentiation

ATF6 is an endoplasmic reticulum (ER) membrane-bound transcription factor that regulates various cellular functions. The purpose of this study was to investigate the role of ATF6 in odontoblast differentiation. Rat tooth germs were isolated, changes in gene expression were evaluated over time, and localization of ATF6 was determined by immunohistochemistry. Human dental pulp cells (HDPCs) were cultured with 50 µg/mL ascorbic acid and 5 mmol/L β-glycerophosphate or 100 ng/mL bone morphogenetic protein 2 to induce differentiation. Translocation of ATF6 was observed by immunofluorescence and confocal microscopy. Overexpression of ATF6 was performed with an adenoviral vector. Matrix mineralization was evaluated by alizarin red staining. Immunoreactivity to anti-ATF6 was observed in the odontoblastic layer of the molar tooth germ, and expressions of ATF6, dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) increased gradually during tooth germ development. When HDPCs were cultured in differentiation media, ATF6, DSPP, and DMP1 expression increased with the expression of unfolded protein response (UPR) markers, BiP and CHOP. Immunofluorescence results showed that ATF6 protein moved from cytoplasm to nucleus when cells were exposed to differentiation media. Notably, overexpression of ATF6 increased DSPP and DMP1 expression, alkaline phosphatase (ALP) activity, and matrix mineralization in HDPC cultures. Inhibition of ATF6 decreased ALP activity and mineralization. These results suggest that ER membrane-bound transcriptional factor ATF6 may be involved in odontoblastic differentiation.

MDM2 E3 ligase-mediated ubiquitination and degradation of HDAC1 in vascular calcification.

Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC.

Cyclic AMP response element-binding protein H (CREBH) mediates the inhibitory actions of tumor necrosis factor α in osteoblast differentiation by stimulating Smad1 Degradation

Background: Severe inflammatory reactions delay wound healing of bone. Results: Tumor necrosis factor α (TNFα) inhibition of osteoblast differentiation is associated with increased cAMP response element-binding protein H (CREBH) and Smurf1 expression. Conclusion: CREBH mediates the inhibitory actions of TNFα in bone regeneration. Significance: CREBH is identified as a new mediator of inflammation-dependent bone degradation and a potential therapeutic target. Endoplasmic reticulum (ER) stress transducers, such as old astrocyte specifically induced substance (OASIS) and activating transcription factor 6 (ATF6), which are induced by bone morphogenetic protein 2 (BMP2), regulate bone formation and osteoblast differentiation. Here, we examined the role of cAMP response element-binding protein H (CREBH), a member of the same family of ER membrane-bound basic leucine zipper (bZIP) transcription factors as OASIS and ATF6, in osteoblast differentiation and bone formation. Proinflammatory cytokine TNFα increased CREBH expression by up-regulating the nuclear factor-κB (NF-κB) signaling pathway in osteoblasts, increased the level of N-terminal fragment of CREBH in the nucleus, and inhibited BMP2 induction of osteoblast specific gene expression. Overexpression of CREBH suppressed BMP2-induced up-regulation of the osteogenic markers runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OC) in MC3T3-E1 cells and primary osteoblasts, as well as BMP2-induced ALP activity and OC protein production. In contrast, knockdown of CREBH attenuated the inhibitory effect of TNFα on BMP2-induced osteoblast differentiation. Mechanistic studies revealed that CREBH increased the expression of Smad ubiquitination regulatory factor 1 (Smurf1), leading to ubiquitin-dependent degradation of Smad1, whereas knockdown of CREBH inhibited TNFα-mediated degradation of Smad1 by Smurf1. Consistent with these in vitro findings, administration of Ad-CREBH inhibited BMP2-induced ectopic and orthotopic bone formation in vivo. Taken together, these results suggest that CREBH is a novel negative regulator of osteoblast differentiation and bone formation.

The Angiopoietin-1 Variant COMP-Ang1 Enhances BMP2-Induced Bone Regeneration with Recruiting Pericytes in Critical Sized Calvarial Defects

Craniofacial bone defects are observed in a variety of clinical situations, and their reconstructions require coordinated coupling between angiogenesis and osteogenesis. In this study, we explored the effects of cartilage oligomeric matrix protein-angiopoietin 1 (COMP-Ang1), a synthetic and soluble variant of angiopoietin 1, on bone morphogenetic protein 2 (BMP2)-induced cranial bone regeneration, and recruitment and osteogenic differentiation of perivascular pericytes. A critical-size calvarial defect was created in the C57BL/6 mouse and COMP-Ang1 and/or BMP2 proteins were delivered into the defects with absorbable collagen sponges. After 3 weeks, bone regeneration was evaluated using micro-computed tomography and histologic examination. Pericyte recruitment into the defects was examined using immunofluorescence staining with anti-NG2 and anti-CD31 antibodies. In vitro recruitment and osteoblastic differentiation of pericyte cells were assessed with Boyden chamber assay, staining of calcified nodules, RT-PCR and Western blot analyses. Combined administration of COMP-Ang1 and BMP2 synergistically enhanced bone repair along with the increased population of CD31 (an endothelial cell marker) and NG2 (a specific marker of pericyte) positive cells. In vitro cultures of pericytes consistently showed that pericyte infiltration into the membrane pore of Boyden chamber was more enhanced by the combination treatment. In addition, the combination further increased the osteoblast-specific gene expression, including bone sialoprotein (BSP), osteocalcin (OCN) and osterix (OSX), phosphorylation of Smad/1/5/8, and mineralized nodule formation. COMP-Ang1 can enhance BMP2-induced cranial bone regeneration with increased pericyte recruitment. Combined delivery of the proteins might be a therapeutic strategy to repair cranial bone damage.

Dysregulated Osteoclastogenesis Is Related to Natural Killer T Cell Dysfunction in Rheumatoid Arthritis

To investigate the role played by natural killer T (NKT) cells in osteoclastogenesis and their effects on inflammatory bone destruction.

Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole.

Recently a submicron particle of biphasic calcium phosphate ceramic (BCP) with through-hole (donut-shaped BCP (d-BCP)) was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing.

Calcitriol enhances fat synthesis factors and calpain activity in co-cultured cells

We have conducted an in vitro experiment to determine whether calcitriol can act as a fat synthesizer and/or meat tenderizer when skeletal muscle cells, adipose tissue, and macrophages are co‐cultured. When co‐cultured, pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) expression increased, whereas decreased anti‐inflammatory cytokine (IL‐10 and IL‐15) expression decreased in both C2C12 and 3T3‐L1 cells. Calcitriol increased reactive oxygen species (ROS) production in the media. While adiponectin gene expression decreased, leptin, resistin, CCAAT‐enhancer‐binding protein‐beta (C/EBP‐β), and peroxisome proliferator‐activated receptor gamma (PPAR‐γ) gene expression was significantly (P < 0.047) increased with calcitriol in 3T3‐L1 cells co‐cultured with two different cell types. Inducible nitric oxide synthase (iNOS) protein levels were also stimulated in the C2C12 and 3T3‐L1 cells, but arginase l was attenuated by calcitriol. Cacitriol highly amplified (P = 0.008) µ‐calpain gene expression in co‐cultured C2C12 cells. The results showed an overall increase in pro‐inflammatory cytokines and a decrease in anti‐inflammatory cytokines of C2C12 and 3T3‐L1 cells with calcitriol in co‐culture systems. µ‐Calpain protein was also augmented in differentiated C2C12 cells with calcitriol. These findings suggest that calcitriol can be used as not only fat synthesizer, but meat tenderizer, in meat‐producing animals.

Betulinic acid synergically enhances BMP2-induced bone formation via stimulating Smad 1/5/8 and p38 pathways

BackgroundHealing of bone defects is a dynamic and orchestrated process that relies on multiple growth factors and cell types. Bone morphogenetic protein 2 (BMP2) is a key growth factor for bone healing, which stimulates mesenchymal stem cells to differentiate into osteoblasts. Betulinic acid (BetA) is a natural pentacyclic triterpenoid from plants. This study aimed to examine combinatory effects of BetA and BMP2 on ectopic bone generation in mice.ResultsIn MC3T3-E1 preosteoblast culture, 10–15 μM of BetA increased the alkaline phosphatase (ALP) activity and expression levels of osteogenic marker genes without the decreased cell viability. In addition, BetA synergistically enhanced BMP2-induced gene expressions and mineralization with the enhancement of phosphorylation of Smad1/5/8 and p38. In an in vivo ectopic bone formation model, combination of BetA (50 μg) and BMP2 (3 μg) resulted in increases in the amount of new bone generation, compared with treatment with BMP2 alone. Histological studies showed that bone generation with cortical and trabecular structures was resulted from the combination of BetA and BMP2.ConclusionBetA can enhance in vivo osteogenic potentials of BMP2, possibly via stimulating Smad 1/5/8 and p38 pathways, and combination of both agents can be considered as a therapeutic strategy for bone diseases.