Impaired breakdown of Herwig's epithelial root sheath disturbs tooth root development.

BACKGROUND: Wnt/ß-catenin signaling plays a variety of roles in both the dental epithelium and mesenchyme at most stages of tooth development. In this study, we verified the roles of Hertwig's epithelial root sheath (HERS) breakdown in tooth root development. This breakdown results in formation of epithelial cell rests of Malassez (ERM). RESULTS: Following induction of ß-catenin stabilization in the epithelium of developing tooth at the moment of HERS breakdown, HERS failed to break down for ERM formation. HERS with stabilized ß-catenin was altered into a multicellular layer enveloping elongated root dentin with higher expression of junctional proteins such as Zo-1 and E-cadherin. Importantly, this impairment of HERS breakdown led to arrest of further root elongation. In addition, the portion of root dentin enveloped by the undissociated HERS remained in a hypomineralized state. The odontoblasts showed ectopically higher expression of pyrophosphate regulators including Ank and Npp1, whereas Tnap expression was unchanged. CONCLUSIONS: Our data suggest that Wnt/ß-catenin signaling is decreased in HERS for ERM formation during root development. Furthermore, ERM formation is important for further elongation and dentin mineralization of the tooth roots. These findings may provide new insight to understand the contribution of ERM to root formation.

Sirt6 Activation Ameliorates Inflammatory Bone Loss in Ligature-Induced Periodontitis in Mice.

Periodontitis is an inflammatory disease caused by microorganisms that induce the destruction of periodontal tissue. Inflamed and damaged tissue produces various inflammatory cytokines, which activate osteoclasts and induce alveolar bone loss and, eventually, tooth loss. Sirt6 expression suppresses inflammation and bone resorption; however, its role in periodontitis remains unclear. We hypothesized that Sirt6 has a protective role in periodontitis. To understand the role of Sirt6 in periodontitis, we compared periodontitis with ligature placement around the maxillary left second molar in 8-week-old control (C57BL/6J) male mice to Sirt6-overexpressing Tg (Sirt6Tg) mice, and we observed the resulting phenotypes using micro-CT. MDL801, a Sirt6 activator, was used as a therapy for periodontitis through oral gavage. Pro-inflammatory cytokines and increased osteoclast numbers were observed in alveolar bone tissue under periodontitis surgery. In the same condition, interestingly, protein levels from Sirt6 were the most downregulated among sirtuins in alveolar bone tissue. Based on micro-CT and CEJ-ABC distance, Sirt6Tg was observed to resist bone loss against ligature-induced periodontitis. Furthermore, the number of osteoclasts was significantly reduced in Sirt6Tg-ligated mice compared with control-ligated mice, although systemic inflammatory cytokines did not change. Consistent with this observation, we confirmed that bone loss was significantly reduced when MDL801, a Sirt6 activator, was included in the ligation mouse model. Our findings demonstrate that Sirt6 activation prevents bone loss against ligature-induced periodontitis. Thus, a Sirt6 activator may provide a new therapeutic approach for periodontitis.

Osteoblastic Wls Ablation Protects Mice from Total Body Irradiation-Induced Impairments in Hematopoiesis and Bone Marrow Microenvironment.

Ionizing irradiation (IR) causes bone marrow (BM) injury, with senescence and impaired self-renewal of hematopoietic stem cells (HSCs), and inhibiting Wnt signaling could enhance hematopoietic regeneration and survival against IR stress. However, the underlying mechanisms by which a Wnt signaling blockade modulates IR-mediated damage of BM HSCs and mesenchymal stem cells (MSCs) are not yet completely understood. We investigated the effects of osteoblastic Wntless (Wls) depletion on total body irradiation (TBI, 5 Gy)-induced impairments in hematopoietic development, MSC function, and the BM microenvironment using conditional Wls knockout mutant mice (Col-Cre;Wlsfl/fl) and their littermate controls (Wlsfl/fl). Osteoblastic Wls ablation itself did not dysregulate BM frequency or hematopoietic development at a young age. Exposure to TBI at 4 weeks of age induced severe oxidative stress and senescence in the BM HSCs of Wlsfl/fl mice but not in those of the Col-Cre;Wlsfl/fl mice. TBI-exposed Wlsfl/fl mice exhibited greater impairments in hematopoietic development, colony formation, and long-term repopulation than TBI-exposed Col-Cre;Wlsfl/fl mice. Transplantation with BM HSCs or whole BM cells derived from the mutant, but not Wlsfl/fl mice, protected against HSC senescence and hematopoietic skewing toward myeloid cells and enhanced survival in recipients of lethal TBI (10 Gy). Unlike the Wlsfl/fl mice, the Col-Cre;Wlsfl/fl mice also showed radioprotection against TBI-mediated MSC senescence, bone mass loss, and delayed body growth. Our results indicate that osteoblastic Wls ablation renders BM-conserved stem cells resistant to TBI-mediated oxidative injuries. Overall, our findings show that inhibiting osteoblastic Wnt signaling promotes hematopoietic radioprotection and regeneration.

Local and Systemic Overexpression of COMP-Ang1 Induces Ang1/Tie2-Related Thrombocytopenia and SDF-1/CXCR4-Dependent Anemia.

While supplemental angiopoietin-1 (Ang1) improves hematopoiesis, excessive Ang1 induces bone marrow (BM) impairment, hematopoietic stem cell (HSC) senescence, and erythropoietic defect. Here, we examined how excessive Ang1 disturbs hematopoiesis and explored whether hematopoietic defects were related to its level using K14-Cre;c-Ang1 and Col2.3-Cre;c-Ang1 transgenic mice that systemically and locally overexpress cartilage oligomeric matrix protein-Ang1, respectively. We also investigated the impacts of Tie2 inhibitor and AMD3100 on hematopoietic development. Transgenic mice exhibited excessive angiogenic phenotypes, but K14-Cre;c-Ang1 mice showed more severe defects in growth and life span with higher presence of Ang1 compared with Col2.3-Cre;c-Ang1 mice. Dissimilar to K14-Cre;c-Ang1 mice, Col2.3-Cre;c-Ang1 mice did not show impaired BM retention or senescence of HSCs, erythropoietic defect, or disruption of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis. However, these mice exhibited a defect in platelet production depending on the expression of Tie2 and globin transcription factor 1 (GATA-1), but not GATA-2, in megakaryocyte progenitor (MP) cells. Treatment with Tie2 inhibitor recovered GATA-1 expression in MP cells and platelet production without changes in circulating RBC in transgenic mice. Consecutive AMD3100 administration not only induced irrecoverable senescence of HSCs but also suppressed formation of RBC, but not platelets, via correlated decreases in number of erythroblasts and their GATA-1 expression in B6 mice. Our results indicate that genetic overexpression of Ang1 impairs hematopoietic development depending on its level, in which megakaryopoiesis is preferentially impaired via activation of Ang1/Tie2 signaling, whereas erythropoietic defect is orchestrated by HSC senescence, inflammation, and disruption of the SDF-1/CXCR4 axis.

Npp1 prevents external tooth root resorption by regulation of cervical cementum integrity.

Tooth roots embedded in the alveolar bone do not typically undergo resorption while the bone continues remodeling in its physiological state. In this study, we analyzed genetically modified mice with the functional inactivation of nucleotide pyrophosphatase 1 (Npp1), encoded by ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). This mutation leads to the formation of ectopic cervical cementum vulnerable to external tooth root resorption. Cementoblasts with the inactivation of Enpp1 extensively expressed non-collagenous matrix proteins enriched with bone sialoprotein (Bsp), dentin matrix protein 1 (Dmp1), and osteopontin (Opn), which have roles in mineralization through nucleation and in cell adhesion through the Arg-Gly-Asp (RGD) motif. In cementoblasts with the inactivation of Enpp1, ß-catenin was significantly activated and induced the expression of these non-collagenous matrix proteins. In addition, adenosine triphosphate (ATP), which is the most preferred substrate of Npp1, accumulated extracellularly and autocrinally induced the expression of the receptor activator of nuclear factor κB ligand (Rankl) in cementoblasts with inactivated Npp1. Consequently, these results strongly suggest that functional Npp1 preserves cervical cementum integrity and supports the anti-resorptive properties of tooth roots through ATP homeostasis in the physiological state of cervical cementum.

Overexpression of COMP-Angiopoietin-1 in K14-Expressing Cells Impairs Hematopoiesis and Disturbs Erythrocyte Maturation.

Numerous studies highlight the potential benefits potentials of supplemental cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) through improved angiogenic effects. However, our recent findings show that excessive overexpression of COMP-Ang1 induces an impaired bone marrow (BM) microenvironment and senescence of hematopoietic stem cells (HSCs). Here, we investigated the underlying mechanisms of how excessive COMP-Ang1 affects the function of BM-conserved stem cells and hematopoiesis using K14-Cre;inducible-COMP-Ang1-transgenic mice. Excessive COMP-Ang1 induced peripheral egression and senescence of BM HSCs and mesenchymal stem cells (MSCs). Excessive COMP-Ang1 also caused abnormal hematopoiesis along with skewed differentiation of HSCs toward myeloid lineage rather than lymphoid lineage. Especially, excessive COMP-Ang1 disturbed late-stage erythroblast maturation, followed by decreased expression of stromal cell-derived factor 1 (SDF-1) and globin transcription factor 1 (GATA-1) and increased levels of superoxide anion and p-p38 kinase. However, transplantation with the mutant-derived BM cells or treatment with rhCOMP-Ang1 protein did not alter the frequency or GATA-1 expression of erythroblasts in recipient mice or in cultured BM cells. Together, our findings suggest that excessive COMP-Ang1 impairs the functions of BM HSCs and MSCs and hematopoietic processes, eventually leading to abnormal erythropoiesis via imbalanced SDF-1/CXCR4 axis and GATA-1 expression rather than Ang1/Tie2 signaling axis alterations.

Cell dynamics in Hertwig's epithelial root sheath are regulated by ß-catenin activity during tooth root development.

ß-catenin, a key mediator of Wnt signaling, plays multiple roles in tooth development. However, the role of ß-catenin in Hertwig's epithelial root sheath (HERS) during root formation remains unclear. In this study, we generated inducible tissue-specific ß-catenin conditional knockout mice (Ctnnb1i∆shh ) to investigate how ß-catenin in HERS affects tooth root development. The inactivation of ß-catenin in HERS led to interrupted root elongation due to premature disruption of HERS. This phenotype was accompanied by reduced cell-cell adhesion and decreased expression of junctional proteins, as well as increased epithelial-to-mesenchymal transition of HERS cells upon ß-catenin depletion. Accordingly, stabilization of ß-catenin in HERS (Catnbi∆shh ) led to the formation of unfragmented HERS and resulted in the failure of HERS dissociation, with increased expression of junctional proteins. Our results suggest that fine control of ß-catenin is important for HERS to guide root formation through regulating its structural integrity.

Osteoblastic Wntless deletion differentially regulates the fate and functions of bone marrow-derived stem cells in relation to age.

Although functional association between Wnt signaling and bone homeostasis has been well described through genetic ablation of Wntless (Wls), the mechanisms of how osteoblastic Wls regulates the fate of bone marrow stromal cells (BMSCs) and hematopoietic stem cells (HSCs) in relation to age are not yet understood. Here, we generated Col2.3-Cre;Wlsfl/fl mice that were free from premature lethality and investigated age-related impacts of osteoblastic Wls deficiency on hematopoiesis, BM microenvironment, and maintenance of BMSCs (also known as BM-derived mesenchymal stem/stromal cells) and HSCs. Ablation of osteoblastic Wls deteriorated BM microenvironment and bone mass accrual along with age-independent effects on functions of BMSCs. Osteoblastic Wls deletion impaired HSC repopulation and progeny with skewing toward myeloid lineage cells only at old stage. As proven by hallmarks of stem cell senescence, osteoblastic Wls ablation differentially induced senescence of BMSCs and HSCs in relation to age without alteration in their BM frequency. Our findings support that deletion of Wls in Col2.3-expressing cells induces senescence of BMSCs and impairs BM microenvironment in age-independent manner. Overall, long-term deterioration in BM microenvironment contributes to age-related HSC senescence with impaired progeny and hematopoiesis, which also suggests possible roles of osteoblastic Wls on the maintenance of BM HSCs.

Genetic overexpression of COMP-Ang1 impairs BM microenvironment and induces senescence of BM HSCs.

Supplemental Angiopoietin 1 (Ang1) exerts its therapeutic potential on microvascular regression-associated diseases, and this potential is linked with the function of hematopoietic stem cells (HSCs). However, the underlying mechanisms of the effect of enhanced angiogenesis on the modulation of HSCs are not yet defined. Here, we generated transgenic mice expressing Cartilage Oligomeric Matrix Protein (COMP)-Ang1 in keratin 14-expressing cells. The mutant animals expressed excessive angiogenic characteristics in the skin and bone marrow (BM) along with redder skin with more numerous and branched vessels compared with their wild-type (WT) littermates. The mutants displayed reduced long bone formation and osteoclast activity than did WT littermates and had fewer CD150+CD48-Lineage-Sca-1+c-Kit+ (LSK) cells in the BM. The mutants also exhibited greater senescence-associated (SA) ß-gal activity, p16INK4a protein expression, and superoxide anion levels in CD150+CD48-LSK cells in the BM. Furthermore, transplantation assay revealed that the mutant-derived LSK cells were inferior to the cells derived from WT littermate in inducing competitive repopulating capacity in the recipients. Collectively, our results demonstrate that persistent and prolonged administration of COMP-Ang1 by inducible transgenic expression mediates excessive angiogenesis in the body and impairs BM microenvironment, eventually leading to senescence of BM HSCs.

Constitutive Activation of Smoothened in the Renal Collecting Ducts Leads to Renal Hypoplasia, Hydronephrosis, and Hydroureter.

Sonic Hedgehog (Shh) signaling plays a major role in and is essential for regulation, patterning, and proliferation during renal development. Smoothened (Smo) plays a pivot role in transducing the Shh-glioma-associated oncogene Kruppel family member. However, the cellular and molecular mechanism underlying the role of sustained Smo activation in postnatal kidney development is still not clearly understood. Using a conditional knockin mouse model that expresses a constitutively activated form of Smo (SmoM2) upon Homeobox-B7-mediated recombination (Hoxb7-Cre), the effects of Shh signaling were determined in postnatal kidney development. SmoM2;Hoxb7-Cre mutant mice showed growth retardation with a reduction of body weight. Constitutive activation of Smo in the renal collecting ducts caused renal hypoplasia, hydronephrosis, and hydroureter. The parenchymal area and glomerular numbers were reduced, but the glomerular density was increased in SmoM2;Hoxb7-Cre mutant mice. The expression of Patched 1, the receptor of Shh and a downstream target gene of the Shh signaling pathway, was highly restricted and it was upregulated in the inner medullary collecting ducts of the kidney. The proliferative cells in the mesenchyme and collecting ducts were decreased in SmoM2;Hoxb7-Cre mutant mice. This study showed for the first time that sustained Smo inhibits postnatal kidney development by suppressing the proliferation of the mesenchyme and medullary collecting ducts in mice.

Maturation of cortical bone suppresses periosteal osteoprogenitor proliferation in a paracrine manner.

Periosteum contains enriched pools of osteogenic progenitors and is highly proliferative, thus giving this tissue a pivotal role in maintaining the diameter of the diaphyseal cortex and in recovery from fractures. Although periosteal proliferation has not been detected in normal bone, intense periosteal proliferation has been observed in pathologic states such as fracture, inflammation, and bone tumors. However, the mechanism by which periosteal osteoprogenitor proliferation is regulated remains poorly understood. To investigate this regulation mechanism, osteoblast/osteocyte-specific conditional knockout mice were developed lacking Smad4 and Osx, two factors that are essential for osteoblast differentiation and matrix mineralization. In Smad4 (Col) and Osx (Col) mice, osteocalcin, Dmp-1, and sclerostin expression were significantly decreased in the cortical bone. Interestingly, although Cre activity was not observed in the periosteum, the proliferation of periosteal osteoprogenitors was enhanced in Smad4 (Col) and Osx (Col) mice, as assessed by 5'-bromo-2'deoxyuridine incorporation and proliferating cell nuclear antigen localization. Since Wnt signaling is a major factor affecting periosteal proliferation, we evaluated Wnt signaling in the periosteum. The expression levels of ß-catenin and Lef-1 were increased in the periosteal osteoprogenitors. Moreover, the mRNA levels of ß-catenin, cyclin D1, Lef-1, and Axin2, all of which are Wnt target genes, were significantly increased in the periosteum of both Smad4 (Col) and Osx (Col) mice. These results indicated that extracellular proteins secreted by mature osteoblasts and osteocytes suppress the proliferation of periosteal osteoprogenitors by blocking Wnt signaling in a paracrine manner. Our data suggest a new concept of periosteal bone healing and periosteal bone formation.

BST2 Mediates Osteoblast Differentiation via the BMP2 Signaling Pathway in Human Alveolar-Derived Bone Marrow Stromal Cells.

The molecular mechanisms controlling the differentiation of bone marrow stromal stem cells into osteoblasts remain largely unknown. In this study, we investigated whether bone marrow stromal antigen 2 (BST2) influences differentiation toward the osteoblasts lineage. BST2 mRNA expression in human alveolar-derived bone marrow stromal cells (hAD-BMSCs) increased during differentiation into osteoblasts. hAD-BMSCs differentiation into osteoblasts and the mRNA expression of the bone-specific markers alkaline phosphatase, collagen type α 1, bone sialoprotein, osteocalcin, and osterix were reduced by BST2 knockdown using siRNA. Furthermore, BST2 knockdown in hAD-BMSCs resulted in decreased RUNX2 mRNA and protein expression. We hypothesized that BST2 is involved in differentiation of into osteoblasts via the BMP2 signaling pathway. Accordingly, we evaluated the mRNA expression levels of BMP2, BMP receptors (BMPR1 and 2), and the downstream signaling molecules SMAD1, SMAD4, and p-SMAD1/5/8 in BST2 knockdown cells. BMP2 expression following the induction of differentiation was significantly lower in BST2 knockdown cells than in cells treated with a non-targeting control siRNA. Similar results were found for the knockdown of the BMP2 receptor- BMPR1A. We also identified significantly lower expression of SMAD1, SMAD4, and p-SMAD1/5/8 in the BST2 knockdown cells than control cells. Our data provide the first evidence that BST2 is involved in the osteogenic differentiation of bone marrow stromal cells via the regulation of the BMP2 signaling pathway.

Brief Report: Consecutive Alendronate Administration-Mediated Inhibition of Osteoclasts Improves Long-Term Engraftment Potential and Stress Resistance of HSCs.

Osteoclasts form a bone marrow (BM) cavity serving as a hematopoietic niche for the maintenance of hematopoietic stem cells (HSCs). However, the role of osteoclasts in the BM has been controversially reported and remains to be further understood. In the present study, we investigated how osteoclasts affect the modulation of hematopoietic stem/progenitor cells in the BM by administering bisphosphate alendronate (ALN) to B6 mice for 21 consecutive days to inhibit osteoclast activity. ALN treatment caused a reduction in the number of tartrate-resistant acid phosphate (TRAP)-positive osteoclast cells and an increase in bone mineral density, particularly in the trabecular zone, but not in the cortical zone of the BM. Osteoclast inhibition caused by ALN treatment decreased mitochondrial reactive oxygen species (ROS) generation and SA-ß-gal activity of CD150+ CD48- Lineage-Sca-1+ c-Kit+ (LSK) cells, eventually leading to an improvement in the engraftment potential and self-renewal activity of HSCs. Moreover, ALN-treated mice exhibited an enhanced resistance of HSCs in response to the genotoxic stress of 5-fluorouracil, as determined by mitochondrial ROS generation, SA-ß-gal activity, and p16INK4a expression in subsets of LSK and CD150+ CD48- LSK cells as well as competitive assay. Collectively, our findings indicate that inhibition of osteoclast activity improves the long-term engraftment potential and stress resistance of HSCs. Stem Cells 2016;34:2601-2607.

Testicular acid phosphatase induces odontoblast differentiation and mineralization.

Odontoblasts differentiate from dental mesenchyme during dentin formation and mineralization. However, the molecular mechanisms controlling odontoblast differentiation remain poorly understood. Here, we show that expression of testicular acid phosphatase (ACPT) is restricted in the early stage of odontoblast differentiation in proliferating dental mesenchymal cells and secretory odontoblasts. ACPT is expressed earlier than tissue-nonspecific alkaline phosphatase (TNAP) and partly overlaps with TNAP in differentiating odontoblasts. In MDPC-23 odontoblastic cells, expression of ACPT appears simultaneously with a decrease in ß-catenin activity and is abolished with the expression of Phex and Dsp. Knockdown of ACPT in MDPC-23 cells stimulates cell proliferation together with an increase in active ß-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active ß-catenin and cyclin D1. Expression of TNAP, Osx, Phex and Dsp is reduced by knockdown of ACPT but is enhanced by ACPT overexpression. When ACPT is blocked with IgG, alkaline phosphatase activity is inhibited but cell proliferation is unchanged regardless of ACPT expression. These findings suggest that ACPT inhibits cell proliferation through ß-catenin-mediated signaling in dental mesenchyme but elicits odontoblast differentiation and mineralization by supplying phosphate during dentin formation. Thus, ACPT might be a novel candidate for inducing odontoblast differentiation and mineralization for dentin regeneration.

Perilipin+ embryonic preadipocytes actively proliferate along growing vasculatures for adipose expansion.

Despite the growing interest in adipose tissue as a therapeutic target of metabolic diseases, the identity of adipocyte precursor cells (preadipocytes) and the formation of adipose tissue during embryonic development are still poorly understood. Here, we clarified the identity and dynamic processes of preadipocytes in mouse white adipose tissue during embryogenesis through direct examination, lineage tracing and culture systems. Surprisingly, we found that lipid-lacking but perilipin(+) or adiponectin(+) proliferating preadipocytes started to emerge at embryonic day 16.5, and these cells underwent active proliferation until birth. Moreover, these preadipocytes resided as clusters and were distributed along growing adipose vasculatures. Importantly, the embryonic preadipocytes exhibited considerable coexpression of stem cell markers, such as CD24, CD29 and PDGFRα, and a small portion of preadipocytes were derived from PDGFRß(+) mural cells, in contrast to the adult preadipocytes present in the stromal vascular fraction. Further analyses with in vitro and ex vivo culture systems revealed a stepwise but dynamic regulation of preadipocyte formation and differentiation during prenatal adipogenesis. To conclude, we unraveled the identity and characteristics of embryonic preadipocytes, which are crucial for the formation and expansion of adipose tissue during embryogenesis.

Local delivery of COMP-angiopoietin 1 accelerates new bone formation in rat calvarial defects.

Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent in bone reconstruction. However, the potential of COMP-Ang1 to regenerate impaired bone and induce new bone formation has not been completely explored. In this study, male Sprague-Dawley rats underwent calvarial defect surgery and divided into two groups: scaffold treatment alone (control group) and COMP-Ang1-impregnated scaffold (COMP-Ang1 group). According to live micro-CT and histological analyses, the COMP-Ang1 group showed greater new bone formation and maturation than did the control both four and eight weeks after surgery. The values of bone volume, bone mineral density, and bone surface were also higher in the COMP-Ang1 group than in the control at the same weeks after surgery. In addition, the delivery of COMP-Ang1 facilitated significantly the expression of osteoblast-specific markers such as runt-related transcription factor 2 (p < 0.001), osterix (p < 0.001), bone morphogenetic protein-2 (p < 0.001), alkaline phosphatase (p < 0.01), osteocalcin (p < 0.001), and type I collagen (p < 0.05) in newly formed bone, compared with the control. Immunohistochemistric assay supported the COMP-Ang1-facilitated induction of bone-specific markers. Furthermore, COMP-Ang1 augmented the mRNA expression of angiogenic factors, especially of platelet endothelial cell adhesion molecule 1, stromal cell-derived factor 1, and Tie-2 in the defect site. Our current findings demonstrate for the first time that a local delivery of recombinant COMP-Ang1 promotes bone formation in calvarial defects, which is coupled with enhanced angiogenesis and chemoattraction.

COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways.

Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

NFI-C regulates osteoblast differentiation via control of osterix expression.

In bone marrow, bone marrow stromal cells (BMSCs) have the capacity to differentiate into osteoblasts and adipocytes. Age-related osteoporosis is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow. In this study, we demonstrate that disruption of nuclear factor I-C (NFI-C) impairs osteoblast differentiation and bone formation, and increases bone marrow adipocytes. Interestingly, NFI-C controls postnatal bone formation but does not influence prenatal bone development. We also found decreased NFI-C expression in osteogenic cells from human osteoporotic patients. Notably, transplantation of Nfic-overexpressing BMSCs stimulates osteoblast differentiation and new bone formation, but inhibits adipocyte differentiation by suppressing peroxisome proliferator-activated receptor gamma expression in Nfic(-/-) mice showing an age-related osteoporosis-like phenotype. Finally, NFI-C directly regulates Osterix expression but acts downstream of the bone morphogenetic protein-2-Runx2 pathway. These results suggest that NFI-C acts as a transcriptional switch in cell fate determination between osteoblast and adipocyte differentiation in BMSCs. Therefore, regulation of NFI-C expression in BMSCs could be a novel therapeutic approach for treating age-related osteoporosis.

The role of PIN1 on odontogenic and adipogenic differentiation in human dental pulp stem cells.

Recently, the involvement of PIN1, a peptidyl-prolyl cis/trans isomerase, has been reported in age-related bone homeostasis and adipogenesis. However, the role of PIN1 during odontogenic and adipogenic differentiation remains to be fully understood, particularly regarding human dental pulp stem cells (HDPSCs). Thus, in the present study, we have investigated the role of PIN1 in odontogenic and adipogenic differentiation of HDPSCs and signaling pathways possibly involved. PIN1 mRNA and protein level were upregulated in a time-dependent manner during adipogenic differentiation, increasing until 1 day of odontogenic induction and then steadily declined during odontogenic differentiation. Treatment of a known PIN1 inhibitor, juglone, significantly increased odontogenic differentiation as confirmed by alkaline phosphatase (ALP) activity, calcium deposition, and mRNAs induction of odontogenic markers [ALP, osteopontin (OPN), osteocalcin (OCN), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP-1)]. On the contrary, adipogenic differentiation was dramatically reduced upon juglone treatment, with concomitant downregulation of lipid droplet accumulation and adipogenic marker genes [peroxisome proliferation-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (AP2)]. In contrast to PIN1 inhibition, the overexpression of PIN1 via adenoviral infection (Ad-PIN1) in HDPSCs inhibited odontogenic differentiation but increased adipogenic differentiation, in which stem cell property markers such as stage-specific embryonic antigen-4 (SSEA-4) and STRO-1 were upregulated during odontogenic differentiation but downregulated in adiopogenic differentiation. Consistently, juglone-mediated inhibition of PIN1 augmented the osteogenic medium (OM)-induced activation of bone morphogenetic protein (BMP), Wnt/ß-catenin, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NF-κB) pathway, which response was reversed by Ad-PIN1. Moreover, juglone blocked the adipogenic induction medium-induced activation of PPARγ, C/EBPα, C/EBPß, ERK, and NF-κB pathways, which was rescued by Ad-PIN1 infection. In summary, the present study shows for the first time that PIN1 acts as an important modulator of odontogenic and adipogenic differentiation of HDPSCs and may have clinical implications for regenerative dentistry.

Fibroblast growth factor-4 enhances proliferation of mouse embryonic stem cells via activation of c-Jun signaling.

Fibroblast growth factor-4 (FGF4) is expressed in embryonic stages and in adult tissues, where it plays critical roles in modulating multiple cellular functions. However, the exact roles of FGF4 on proliferation and differentiation of embryonic stem cells (ESCs) are not completely understood. Exogenous addition of FGF4 stimulated proliferation of mouse ESCs (mESCs), as proven by the increases in DNA synthesis and cell cycle regulatory protein induction. These increases were almost completely inhibited by pre-treating cells with anti-FGF4 antibody. FGF4 also activated c-Jun N-terminal kinase (JNK) and extracellular-signal regulated kinase (ERK) signaling, but not p38 kinase. Blockage of JNK signaling by SP600125 or by transfection with its specific siRNA significantly inhibited FGF4-stimulated cell proliferation through the suppression of c-Jun induction and activator protein-1 (AP-1) activity. However, ERK or p38 kinase inhibitor did not affect FGF4-stimulated proliferation in mESCs. FGF4 suppressed osteogenic differentiation of mESCs by inhibiting expression of transcription factors involved in bone formation. Further, exogenous FGF4 addition stimulated proliferation of human periodontal ligament stem cells (hPDLSCs) and bone marrow mesenchymal stem cells (BMMSCs) via activation of ERK signaling. FGF4 also augmented mineralization of hPDLSCs, but not of BMMSCs. Collectively, it is suggested that FGF4 triggers proliferation of stem cells by activating MAPK-mediated signaling, while it affects differently osteogenic differentiation according to the origins of stem cells.