Peptidyl-prolyl cis-trans isomerase NIMA interacting 1 regulates skeletal muscle fusion through structural modification of Smad3 in the linker region.

Myoblast fusion is critical for muscle growth, regeneration, and repair. We previously reported that the enzyme peptidyl-prolyl cis-trans isomerase NIMA interacting 1 (Pin1) is involved in osteoclast fusion. The objective of this study was to investigate the possibility that Pin1 also inhibits myoblast fusion. Here, we show the increased number of nuclei in the Pin1+/- mice muscle fiber compared to that in wild-type mice. Moreover, we show that low dose of the Pin1 inhibitor dipentamethylene thiuram monosulfide treatment caused enhanced fusion in C2C12 cells. The R-Smads are well-known mediators of muscle hypertrophy and hyperplasia as well as being substrates of Pin1. We found that Pin1 is crucial for maintaining the stability of Smad3 (homologues of the Drosophila protein, mothers against decapentaplegic (Mad) and the Caenorhabditis elegans protein Sma). Our results show that serine 204 within Smad3 is the key Pin1-binding site during inhibition of myoblast fusion and that both the transforming growth factor-ß receptor and extracellular signal-regulated kinase (ERK)-mediated phosphorylation are required for the interaction of Pin1 with Smad3. These findings suggest that a precise level of Pin1 activity is essential for regulating myoblast fusion during myogenesis and muscle regeneration.

Blood-testis barrier integrity depends on Pin1 expression in Sertoli cells.

The conformation and function of a subset of serine and threonine-phosphorylated proteins are regulated by the prolyl isomerase Pin1 through isomerization of phosphorylated Ser/Thr-Pro bonds. Pin1 is intensely expressed in Sertoli cells, but its function in this post mitotic cell remains unclear. Our aim was to investigate the role of Pin1 in the Sertoli cells. Lack of Pin1 caused disruption of the blood-testis barrier. We next investigated if the activin pathways in the Sertoli cells were affected by lack of Pin1 through immunostaining for Smad3 protein in testis tissue. Indeed, lack of Pin1 caused reduced Smad3 expression in the testis tissue, as well as a reduction in the level of N-Cadherin, a known target of Smad3. Pin1-/- testes express Sertoli cell marker mRNAs in a pattern similar to that seen in Smad3+/- mice, except for an increase in Wt1 expression. The resulting dysregulation of N-Cadherin, connexin 43, and Wt1 targets caused by lack of Pin1 might affect the mesenchymal-epithelial balance in the Sertoli cells and perturb the blood-testis barrier. The effect of Pin1 dosage in Sertoli cells might be useful in the study of toxicant-mediated infertility, gonadal cancer, and for designing male contraceptives.

Efficiency data of intracellular recombinant protein delivery using cationic lipid coated silk fibroin particle.

This article presents data related to the research article "Fibroin particle-supported cationic lipid layers for highly efficient intracellular protein delivery" and focuses on the delivery efficiency aspects of the fibroin particle-cationic lipid complex (Fibroplex), including its fabrication and the intracellular delivery to the mouse skin tissue. We introduced a stable lipid-particle complex called "Fibroplex", formed by loading cargo protein onto a silk fibroin spherical particle core complexed with cationic liposomes to address the intracellular recombinant protein delivery. This system exhibits cationic charge, which is advantageous for cellular uptake. The particle core is loaded with the cargo protein with high efficiency and shows long-term release in serum environment. Fibroplex can be formed simply by mixing the particle core and cationic liposome, and this spontaneous interaction does not cause any detrimental effects on the function of cargo proteins. Lipid-particle complex structure is stable over 10 days in the serum at 37 °C. Fibroplex was delivered at high efficiency to a wide variety of cells, including cancer cells and primary cell-lines. Also, Fibroplex loaded with two types of cargo successfully introduced them into the cytoplasm. Furthermore, Fibroplex shows successful intracellular delivery when injected with various cargo proteins such as GFP, HRP and Tyrosinase into mouse skin tissue as well as in vitro. The highlights of this article include: (1) Data for fabrication procedure of Fibroplex, (2) loading capacity, surface charge changes of Fibroplex, and (3) Intracellular delivery aspects of Fibroin in vitro and vivo.

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. © 2017 American Society for Bone and Mineral Research.

Fibroin particle-supported cationic lipid layers for highly efficient intracellular protein delivery.

Directly delivering therapeutic proteins into cells has promise as an intervention without side effects for protein deficiencies caused by genetic defects. However, as negatively charged macromolecules, proteins require carriers for achieving cellular uptake and maintaining their activity in the cytoplasm. The biodegradable natural polymer silk fibroin has demonstrated outstanding advantages as a protein drug scaffold in vitro and in vivo, but its usage has been limited in the extracellular space because of its negatively charged character. Here, we present an intracellular protein delivery system based on fibroin particles coated with cationic lipid layers, denoted as Fibroplex, the surface charge of which can be modulated. Fibroplex showed higher delivery efficiency than conventional delivery methods as well as long-term cargo release in the cytoplasm without toxicity. Furthermore, in vivo experiments showed that Fibroplex efficiently delivered tyrosinase and horseradish peroxidase, which led to hyper-pigmentation and tumor regression, respectively, suggesting its potential for therapeutic protein applications in hereditary diseases or cancer.

Pin1, the Master Orchestrator of Bone Cell Differentiation.

Pin1 is an enzyme that specifically recognizes the peptide bond between phosphorylated serine or threonine (pS/pT-P) and proline. This recognition causes a conformational change of its substrate, which further regulates downstream signaling. Pin1-/- mice show developmental bone defects and reduced mineralization. Pin1 targets RUNX2 (Runt-Related Transcription Factor 2), SMAD1/5, and ß-catenin in the FGF, BMP, and WNT pathways, respectively. Pin1 has multiple roles in the crosstalk between different anabolic bone signaling pathways. For example, it controls different aspects of osteoblastogenesis and increases the transcriptional activity of Runx2, both directly and indirectly. Pin1 also influences osteoclastogenesis at different stages by targeting PU.1 (Purine-rich nucleic acid binding protein 1), C-FOS, and DC-STAMP. The phenotype of Pin1-/- mice has led to the recent identification of multiple roles of Pin1 in different molecular pathways in bone cells. These roles suggest that Pin1 can be utilized as an efficient drug target in congenital and acquired bone diseases. J. Cell. Physiol. 232: 2339-2347, 2017. © 2016 Wiley Periodicals, Inc.

Direct Delivery of Recombinant Pin1 Protein Rescued Osteoblast Differentiation of Pin1-Deficient Cells.

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 ß-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.

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.

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

The canonical Wnt signaling pathway, in which ß-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 ß-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear ß-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 ß-catenin in the nucleus. The isomerized ß-catenin could not bind to nuclear adenomatous polyposis coli, which drives ß-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of ß-catenin in the nucleus and might explain the decrease of ß-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate ß-catenin-mediated osteogenesis.

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.

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.

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.

Epigenetic modifications and canonical wingless/int-1 class (WNT) signaling enable trans-differentiation of nonosteogenic cells into osteoblasts.

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.

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

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-mediated prolyl isomerization of Runx1 affects PU.1 expression in pre-monocytes.

Regulation of the hematopoietic transcription factor PU.1, a member of the ETS family, plays a critical role in the development of blood cells and in leukemia. The dosage of PU.1 has been shown to cause a shift in myelomonocytic progenitor fate. Pin1 is a unique substrate-specific enzyme that can isomerize phospho-Ser/Thr-Pro peptide bonds, accelerating the conformational change in its substrates between a cis and a trans form. Such activity has been demonstrated to be a tightly controlled mechanism regulating a wide variety of protein functions under both normal physiological and pathological conditions. We have previously reported that a conformational change in Runx2 induced by Pin1 is essential for its function in osteogenesis in vitro and in vivo. In this study, we show that the Pin1-mediated conformational change in Runx1 enhances its acetylation and stabilization and, consequently, enhances its transacting activity. The increased acetylation of Runx1 represses PU.1 transcription in pre-monocytes. Conversely, the lack of (or the inhibition of) Pin1 increases PU.1 transcription in vitro and in vivo in pre-monocytes and in the spleen tissue. Pin1 KO mice have an increased CD11b(+) /F4/80(+) cell population and F4/80 protein expression in spleen. From our data, we can conclude that the conformational change in Runx1 induced by Pin1 represses PU.1 transcription in pre-monocytes and influences the commitment to the monocyte lineage. The dosage of PU.1 is a crucial factor in acute myeloid leukemia (AML), and Pin1 may thus be a useful target for controlling PU.1-dependent hematopoiesis, as well as leukemogenesis.

Pin1-mediated Runx2 modification is critical for skeletal development.

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.

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 ß-catenin with siRNA completely reversed Wnt3a-stimulated Mepe expression. The Mepe mRNA expression level was increased by overexpression of ß-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 ß-catenin and Lef-1 and indirectly through the activation of a Bmp-2 autocrine loop.

Functional characterization of a novel FGFR2 mutation, E731K, in craniosynostosis.

Craniosynostosis is a condition in which some or all of the sutures in the skull of an infant close prematurely. Fibroblast growth factor receptor 2 (FGFR2) mutations are a well-known cause of craniosynostosis. Many syndromes that comprise craniosynostosis, such as Apert syndrome, Crouzon syndrome, and Pfeiffer syndrome, have one of the phenotypes that have been reported in FGFR2 mutant patients. FGFRs have been reported in four types (FGFR1-4), and upon binding with FGF ligands, signal transduction occurs inside of cells. Activated FGFR stimulates an osteogenic master transcription factor, Runx2, through the MAP kinase and PKC pathways. We obtained a genetic analysis of six Korean patients who have craniosynostosis as a phenotype. All of the patients had at least one mutation in the FGFR2 gene; five of those mutations have already been reported elsewhere, while one mutation is novel and was hypothesized to lead to Apert syndrome. In this study, we reported and functionally analyzed a novel mutation of the FGFR2 gene found in a craniosynostosis patient, E731K. The mutation is in the 2nd tyrosine kinase domain in the C-terminal cytoplasmic region of the molecule. The mutation caused an enhanced phosphorylation of the FGFR2(E731K) and ERK-MAP kinase, the stimulation of transcriptional activity of Runx2, and consequently, the enhancement of osteogenic marker gene expression. We conclude that the substitution of E731K in FGFR2 is a novel mutation that resulted in a constitutive activation of the receptor and ultimately resulted in premature suture obliteration.

Crosstalk between nuclear factor I-C and transforming growth factor-ß1 signaling regulates odontoblast differentiation and homeostasis.

Transforming growth factor-ß1 (TGF-ß1) signaling plays a key role in vertebrate development, homeostasis, and disease. Nuclear factor I-C (NFI-C) has been implicated in TGF-ß1 signaling, extracellular matrix gene transcription, and tooth root development. However, the functional relationship between NFI-C and TGF-ß1 signaling remains uncharacterized. The purpose of this study was to identify the molecular interactions between NFI-C and TGF-ß1 signaling in mouse odontoblasts. Real-time polymerase chain reaction and western analysis demonstrated that NFI-C expression levels were inversely proportional to levels of TGF-ß1 signaling molecules during in vitro odontoblast differentiation. Western blot and immunofluorescence results showed that NFI-C was significantly degraded after TGF-ß1 addition in odontoblasts, and the formation of the Smad3 complex was essential for NFI-C degradation. Additionally, ubiquitination assay results showed that Smurf1 and Smurf2 induced NFI-C degradation and polyubiquitination in a TGF-ß1-dependent manner. Both kinase and in vitro binding assays revealed that the interaction between NFI-C and Smurf1/Smurf2 requires the activation of the mitogen-activated protein kinase pathway by TGF-ß1. Moreover, degradation of NFI-C induced by TGF-ß1 occurred generally in cell types other than odontoblasts in normal human breast epithelial cells. In contrast, NFI-C induced dephosphorylation of p-Smad2/3. These results show that crosstalk between NFI-C and TGF-ß1 signaling regulates cell differentiation and homeostatic processes in odontoblasts, which might constitute a common cellular mechanism.

Suppression of Runx2 protein degradation by fibrous engineered matrix.

The fibre structure of engineered matrix that mimic the morphology of type I collagen has exhibited good biological performance for bone regeneration. However, the mechanism by which synthetic fibres promote osteoblast differentiation has yet to be determined. In this study, we demonstrate that fibre structure of an engineered matrix suppresses the degradation of Runx2, a master transcription factor that can turn on to osteoblast differentiation. MC3T3-E1 pre-osteoblasts grown on a fibrous collagen matrix sustained a higher level of Runx2 protein than those on tissue culture dishes or on a collagenase-treated, non-fibrous collagen matrix. The ubiquitin-dependent degradation of Runx2 was profoundly decreased in cells grown on the fibrous collagen matrix. The forced expression of Smurf1, an ubiquitin ligase responsible for Runx2 degradation, abrogated the collagen fibre-induced increase of Runx2. We also prepared a polystyrene fibre matrix, and confirmed that the fibre matrix stabilised the Runx2 protein in MC3T3-E1. Furthermore, we genetically modified C2C12 myoblasts with Runx2, cultured the cells on polystyrene fibre matrix, and observed that the fibre matrix stabilised and sustained exogenous Runx2, which led to the promotion of osteoblast differentiation. Our findings in this study provide evidence that the fibre structure of an engineered matrix contributes to osteoblast differentiation by stabilising the Runx2 protein.