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Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.

Nanofibrous engineered matrices have significant potential in cellular differentiation and tissue regeneration. Stem cells require specific extracellular signals that lead to the induction of different lineages. However, the mechanisms by which the nanofibrous matrix promotes mesenchymal stem cell (MSC) differentiation are largely unknown. Here, we investigated the mechanisms that underlie nanofibrous matrix-induced odontoblastic differentiation of human dental pulp MSCs (DP-MSCs). An electrospun polystyrene nanofibrous (PSF) matrix was prepared, and the cell responses to the PSF matrix were assessed in comparison with those on conventional tissue culture dishes. The PSF matrix promoted the expression of Wnt3a, Wnt5a, Wnt10a, BMP2, BMP4, and BMP7 in the DP-MSCs, concomitant with the induction of odontoblast/osteoblast differentiation markers, dentin sialophosphoprotein (DSPP), osteocalcin, and bone sialoprotein, whose levels were further enhanced by treatment with recombinant Wnt3a. The DP-MSCs cultured on the PSF matrix also exhibited a high alkaline phosphatase activity and intense Alizarin Red staining, indicating that the PSF matrix promotes odontoblast differentiation. Besides inducing the expression of Wnt3a, the PSF matrix maintained high levels of β-catenin protein and enhanced its translocation to the nucleus, leading to its transcriptional activity. Forced expression of LEF1 or treatments with LiCl further enhanced the DSPP expression. Blocking the Wnt3a-initiated signaling abrogated the PSF-induced DSPP expression. Furthermore, the cells on the PSF matrix increased the DSPP promoter activity. The β-catenin complex was bound to the conserved motifs on the DSPP promoter dictating its transcription. Transplantations of the preodontoblast-seeded PSF matrix to the subcutaneous tissues of nude mice confirmed the association of the PSF matrix with the Wnt3a and DSPP expressions in vivo. Taken together, these results demonstrate the nanofibrous engineered matrix strongly supports odontoblastic differentiation of DP-MSCs by enhancing Wnt/β-catenin signaling.

Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine-proline or phosphothreonine-proline motifs of several proteins. We reported that Pin1 plays a critical role in the fate determination of Smad1/5, Runx2, and β-catenin that are indispensable nuclear proteins for osteoblast differentiation. Though several chemical inhibitors has been discovered for Pin1, no activator has been reported as of yet. In this study, we directly introduced recombinant Pin1 protein successfully into the cytoplasm via fibroin nanoparticle encapsulated in cationic lipid. This nanoparticle-lipid complex delivered its cargo with a high efficiency and a low cytotoxicity. Direct delivery of Pin1 leads to increased Runx2 and Smad signaling and resulted in recovery of the osteogenic marker genes expression and the deposition of mineral in Pin1-deficient cells. These result indicated that a direct Pin1 protein delivery method could be a potential therapeutics for the osteopenic diseases. J. Cell. Physiol. 232: 2798-2805, 2017. © 2016 Wiley Periodicals, Inc.

Bone regeneration has been a challenge for both researchers and clinicians. In the field of tissue engineering, much effort has been made to identify cell sources including stem cells. The present study aimed to induce trans-differentiation from adipocytes to osteoblasts using epigenetic modifiers; 5-aza-dC and/or trichostatin-A (TSA). 3 T3-L1 preadipocytes were treated with TSA (100 nM) and then with Wnt3a (50 ng/ml). Microscopic observation showed trans-differentiated cell morphology. Methylation-specific PCR and immunoblotting were performed to analyze the DNA methylation and histone acetylation patterns. The gene expression was determined by real-time PCR. Based on these in vitro experiments, in vivo mouse experiments supplemented the possibility of trans-differentiation by epigenetic modification. TSA induced the acetylation of lysine9 on histone H3, and a sequential Wnt3a treatment stimulated the expression of bone marker genes in adipocytes, suppressing adipogenesis and stimulating osteogenesis. Furthermore, TSA induced DNA hypomethylation, and a combined treatment with TSA and 5-aza-dC showed a synergistic effect in epigenetic modifications. The number of adipocytes and DNA methylation patterns of old (15 months) and young (6 weeks) mice were significantly different, and TSA and sequential Wnt3a treatments increased bone formation in the old mice. Collectively, our results confirmed cell trans-differentiation via epigenetic modifications and osteogenic signaling from adipocytes to osteoblasts for the bone regeneration in vitro and in vivo, and indicated that histone acetylation could induce DNA hypomethylation, enhancing the chance of trans-differentiation.

It is widely accepted that sandblasted/large-grit/acid-etched (SLA) surfaces of titanium (Ti) have a higher osteogenic potential than machined ones. However, most studies focused on differential gene expression without elucidating the underlying mechanism for this difference. The aim of this study was to evaluate how the surface roughness of dental Ti implants affects their osteogenic potential. Mouse preosteoblast MC3T3-E1 cells were seeded on machined and SLA Ti discs. The cellular activities of the discs were analyzed using confocal laser scanning microscopy, proliferation assays, and real-time polymerase chain reaction (PCR). DNA methylation was evaluated using a methylation-specific PCR. The cell morphology was slightly different between the two types of surfaces. While cellular proliferation was slightly greater on the machined surfaces, the osteogenic response of the SLA surfaces was superior, and they showed increased alkaline phosphatase (Alp) activity and higher bone marker gene expression levels (Type I collagen, Alp, and osteocalcin). The degree of DNA methylation on the Alp gene was lower on the SLA surfaces than on the machined surfaces. DNA methyltransferase inhibitor stimulated the Alp gene expression on the machined surfaces, similar to the SLA surfaces. The superior osteogenic potential of the SLA surfaces can be attributed to a different epigenetic landscape, specifically, the DNA methylation of Alp genes. This finding offers novel insights into epigenetics to supplement genetics and raises the possibility of using epidrugs as potential therapeutic targets to enhance osteogenesis on implant surfaces.

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.

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.

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.

Runx2 is the master transcription factor for bone formation. Haploinsufficiency of RUNX2 is the genetic cause of cleidocranial dysplasia (CCD) that is characterized by hypoplastic clavicles and open fontanels. In this study, we found that Pin1, peptidyl prolyl cis-trans isomerase, is a critical regulator of Runx2 in vivo and in vitro. Pin1 mutant mice developed CCD-like phenotypes with hypoplastic clavicles and open fontanels as found in the Runx2+/− mice. In addition Runx2 protein level was significantly reduced in Pin1 mutant mice. Moreover Pin1 directly interacts with the Runx2 protein in a phosphorylation-dependent manner and subsequently stabilizes Runx2 protein. In the absence of Pin1, Runx2 is rapidly degraded by the ubiquitin-dependent protein degradation pathway. However, Pin1 overexpression strongly attenuated uniquitin-dependent Runx2 degradation. Collectively conformational change of Runx2 by Pin1 is essential for its protein stability and possibly enhances the level of active Runx2 in vivo.

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.

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.

Mesenchymal cells alter and retain their phenotype during skeletal development through activation or suppression of signaling pathways. For example, we have shown that Wnt3a only stimulates osteoblast differentiation in cells with intrinsic osteogenic potential (e.g. MC3T3-E1 pre-osteoblasts) and not in fat cell precursors or fibroblasts (3T3-L1 pre-adipocytes or NIH3T3 fibroblasts, respectively). Wnt3a promotes osteogenesis in part by stimulating autocrine production of the osteoinductive ligand Bmp2. Here, we show that the promoter regions of the genes for Bmp2 and the osteoblast marker Alp are epigenetically locked to prevent their expression in nonosteogenic cells. Both genes have conserved CpG islands that exhibit increased CpG methylation, as well as decreased acetylation and increased methylation of histone H3 lysine 9 (H3-K9) specifically in nonosteogenic cells. Treatment of pre-adipocytes or fibroblasts with the CpG-demethylating agent 5′-aza-2′-deoxycytidine or the histone deacetylase inhibitor trichostatin-A renders Bmp2 and Alp responsive to Wnt3a. Hence, drug-induced epigenetic activation of Bmp2 gene expression contributes to Wnt3a-mediated direct trans-differentiation of pre-adipocytes or fibroblasts into osteoblasts. We propose that direct conversion of nonosteogenic cells into osteoblastic cell types without inducing pluripotency may improve prospects for novel epigenetic therapies to treat skeletal afflictions.

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.

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.

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.