Han-Sol Bae

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.

Pin1 regulates osteoclast fusion through suppression of the master regulator of cell fusion DC-STAMP.

Cell fusion is a fundamental biological event that is essential for the development of multinucleated cells such as osteoclasts. Fusion failure leads to the accumulation of dense bone such as in osteopetrosis, demonstrating the importance of fusion in osteoclast maturity and bone remodeling. In a recent study, we reported that Pin1 plays a role in the regulation of bone formation and Runx2 regulation. In this study, we explored the role of Pin1 in osteoclast formation and bone resorption. Pin1 null mice have low bone mass and increased TRAP staining in histological sections of long bones, compared to Pin1 wild‐type mice. In vitro osteoclast forming assays with bone marrow‐derived monocyte/macrophage revealed that Pin1‐deficient osteoclasts are larger than wild‐type osteoclasts and have higher nuclei numbers, indicating greater extent of fusion. Pin1 deficiency also highly enhanced foreign body giant cell formation both in vitro and in vivo. Among the known fusion proteins, only DC‐STAMP was significantly increased in Pin1−/− osteoclasts. Immunohistochemistry showed that DC‐STAMP expression was also significantly increased in tibial metaphysis of Pin1 KO mice. We found that Pin1 binds and isomerizes DC‐STAMP and affects its expression levels and localization at the plasma membrane. Taken together, our data indicate that Pin1 is a determinant of bone mass through the regulation of the osteoclast fusion protein DC‐STAMP. The identification of Pin1 as a factor involved in cell fusion contributes to the understanding of osteoclast‐associated diseases, including osteoporosis, and opens new avenues for therapeutic targets. J. Cell. Physiol. 229: 2166–2174, 2014.

Genome-wide identification of target genes for miR-204 and miR-211 identifies their proliferation stimulatory role in breast cancer cells

MiR-204 and miR-211 (miR-204/211) share the same seed site sequence, targeting many of the same genes. Their role in cancer development remains controversial, as both cell proliferative and suppressive effects have been identified. This study aimed to address the relationship between the two structurally similar microRNAs (miRs) by examining their target genes in depth as well as to reveal their contribution in breast cancer cells. Genome-wide pathway analysis with the dysregulated genes after overexpression of either of the two miRs in MCF-7 breast cancer cell identified the “Cancer”- and “Cell signaling”-related pathway as the top pathway for miR-204 and miR-211, respectively. The majority of the target genes for both miRs notably comprised ones that have been characterized to drive cells anti-tumorigenic. Accordingly, the miRs induced the proliferation of MCF-7 and MDA-MB-231 cells, judged by cell proliferation as well as colony forming assay. Tumor suppressors, MX1 and TXNIP, were proven to be direct targets of the miRs. In addition, a high association was observed between miR-204 and miR-211 expression in breast cancer tissue. Our results indicate that miR-204/211 serve to increase cell proliferation at least in MCF-7 and MDA-MB-231 breast cancer cells by downregulating tumor suppressor genes.

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.

Epigenetic silencing of miR-19a-3p by cold atmospheric plasma contributes to proliferation inhibition of the MCF-7 breast cancer cell

Cold atmospheric plasma (CAP) has been proposed as a useful cancer treatment option after showing higher induction of cell death in cancer cells than in normal cells. Although a few studies have contributed to elucidating the molecular mechanism by which CAP differentially inhibits cancer cell proliferation, no results are yet to be reported related to microRNA (miR). In this study, miR-19a-3p (miR-19a) was identified as a mediator of the cell proliferation-inhibitory effect of CAP in the MCF-7 breast cancer cell. CAP treatment of MCF-7 induced hypermethylation at the promoter CpG sites and downregulation of miR-19a, which was known as an oncomiR. The overexpression of miR-19a in MCF-7 increased cell proliferation, and CAP deteriorated the effect. The target genes of miR-19a, such as ABCA1 and PTEN, that had been suppressed by miR recovered their expression through CAP treatment. In addition, an inhibitor of reactive oxygen species that is produced by CAP suppressed the effect of CAP on cell proliferation. Taken together, the present study, to the best of authors’ knowledge, is the first to identify the involvement of a miR, which is dysregulated by the CAP and results in the anti-proliferation effect of CAP on cancer cells.

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.

Biomimetic approach to stimulate osteogenesis on titanium implant surfaces using fibronectin derived oligopeptide.

Our previous studies demonstrated that a recombinant fibronectin (FN)-derived oligopeptide that we named F20 stimulated osteoblast adhesion, proliferation, and differentiation in vitro and in vivo. In the present study, we used a synthetic oligopeptide and investigated the osteogenic potential of F20 coating on titanium discs, to stimulate superior osseointegration for dental implant surface modification. Surface characteristic analysis of titanium was performed by confocal laser scanning microscopy (CLSM) observation. Synthetic F20 was coated onto the machined or SLA titanium discs by an adsorption procedure. ST2 cells were seeded on the titanium discs. We evaluated cell adhesion with SEM and CLSM observation, cell proliferation with picogreen assay, and osteoblast differentiation with real-time PCR, ALP activity assay, immunoblot assay and ALP staining. FITC-labeled F20 coating on the discs was detected by fluorescence, showing good F20 adsorption and different coating patterns according to the surface roughness. In the SEM and CLSM observations, cells were well attached on the machined surface and greater stress fiber formation was seen on discs coated with F20 than on other discs. F20 stimulated cellular proliferation, as well as osteoblast differentiation through the extracellular signalregulated kinase (Erk) signaling pathway. These cellular responses to F20 were slightly better on the machined titanium surface than the SLA surface. These results suggest that F20 promotes osteogenesis through the Erk pathway and is a suitable biomolecule for surface modification of dental implants for improved osseointegration.

A Novel FGFR2 Mutation in Tyrosine Kinase II Domain, L617F, in Crouzon Syndrome

The purposes of this study were to find a novel mutation of FGFR2 in Korean Crouzon syndrome patients and to identify the functional consequences of this mutation. The samples consisted of 16 Crouzon patients. Peripheral venous blood was collected from the patients. FGFR2 mutation screening was performed by direct PCR sequencing of all exons and part of the introns. Restriction fragment length polymorphism (RFLP) analysis was performed to confirm the novel mutation. For functional studies, we performed luciferase assay for Runx2 transcriptional activity, real‐time PCR for the bone markers (osteocalcin and alkaline phosphatase), and Western blot for phosphorylated FGFR2 and ERK1/2‐MAPK protein. Among 16 patients, 10 showed FGFR2 mutations that had already been reported elsewhere. A novel FGFR2 mutation associated with tyrosine kinase II (TK‐II) domain, L617F, was found in one Crouzon syndrome patient by direct PCR sequencing. Presence of this mutation was confirmed using RFLP analysis. Runx2 transcriptional activity and expression of osteocalcin and alkaline phosphatase significantly increased in L617F‐transfected cells compared to wild‐type cells. FGFR2 autophosphorylation in L617F‐transfected cells increased in 1% serum, but ERK1/2‐MAPK protein was not activated. The FGFR2‐L617F mutation associated with the TK domain is potentially related to premature suture closure in Crouzon syndrome patient. J. Cell. Biochem. 115: 102–110, 2014.

Direct Gingival Fibroblast/Osteoblast Transdifferentiation via Epigenetics

Alveolar bone resorption caused by trauma or periodontal diseases has represented a challenge for both dental clinicians and researchers. In this study, we evaluate the osteogenic potential of human gingival fibroblasts (HGFs) through a direct transdifferentiation from HGFs to functional osteoblasts via epigenetic modification and osteogenic signaling with bone morphogenetic protein 2 (BMP2) in vitro and in vivo. HGF treatment with 5-aza-2′-deoxycytidine (5-aza-dC) induced demethylation in the hypermethylated CpG islands of the osteogenic lineage marker genes RUNX2 and ALP, and subsequent BMP2 treatment successfully drove the fibroblasts to the osteoblasts’ lineage. Cell morphological changes viewed under microscopy and alkaline phosphatase (ALP) and alizarin red S (ARS) staining confirmed the osteoblastic change mediated by epigenetic modification as did real-time polymerase chain reaction (PCR), methylation-specific PCR (MSP), and chromatin immunoprecipitation (ChIP) assay, which demonstrated the altered methylation patterns in the RUNX2 and ALP promoter regions and their effect on gene expression. Furthermore, micro–computed tomography (CT) analysis of in vivo mouse cell transplantation experiments showed high-density signal in the epigenetically modified HGF group; in addition, a significant amount of bone formation was observed in the transplanted material using hematoxylin and eosin (H&E) staining as well. Collectively, our results indicate that epigenetic modification permits the direct programming of HGFs into functional osteoblasts, suggesting that this approach might open a novel therapeutic avenue in alveolar bone regeneration.