Runt-related Protein Research Articles

IGF-1 Induces Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells by Promoting SOX4 via the MAPK/ERK Pathway.

Tissue engineering envisions functional substitute creation for damaged tissues. Insulin-like growth factor-1 (IGF-1) plays roles in bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation (OD), and we investigated its specific mechanism. BMSCs were cultured and OD was induced. Surface antigens (CD105, CD90, CD44, CD45, CD34) were identified by flow cytometry. Adipogenic, chondrogenic, and osteogenic differentiation abilities of BMSCs were observed. BMSCs were cultured in osteogenic medium containing 80 ng/mL IGF-1 for 3 weeks. Alkaline phosphatase activity, calcification level, osteogenic factor (runt related protein 2 [RUNX2], osteocalcin [OCN], osterix [OSX]), total (t-) ERK1/2 and phosphorylated- (p-) ERK1/2 levels, and SRY-related high-mobility-group box 4 (SOX4) levels were assessed by alkaline phosphatase staining and Alizarin Red staining, Western blot, and reverse transcription-quantitative polymerase chain reaction. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway inhibitor (PD98059) was used to inhibit the MAPK/ERK pathway in IGF-1-treated BMSCs. Small interfering-SOX4 was transfected into BMSCs to down-regulate SOX4. IGF-1 increased alkaline phosphatase activity, cell calcification, and osteogenic factor (RUNX2, OCN, OSX) levels in BMSCs, indicating that IGF-1 induced rat BMSC OD. SOX4, and p-ERK1/2 and t-ERK1/2 levels were elevated in IGF-1-induced BMSCs, which were annulled by PD98059. PD98059 partly averted IGF-1-induced rat BMSC OD. SOX4 levels, alkaline phosphatase activity, cell calcification, and osteogenic factor (RUNX2, OCN, OSX) levels were reduced after SOX4 down-regulation, showing that downregulation of SOX4 averted the effect of IGF-1 on inducing rat BMSC OD. IGF-1 induced rat BMSC OD by stimulating SOX4 via the MAPK/ERK pathway.

RUNX Family as a Promising Biomarker and a Therapeutic Target in Bone Cancers: A Review on Its Molecular Mechanism(s) behind Tumorigenesis.

The transcription factor runt-related protein (RUNX) family is the major transcription factor responsible for the formation of osteoblasts from bone marrow mesenchymal stem cells, which are involved in bone formation. Accumulating evidence implicates the RUNX family for its role in tumor biology and cancer progression. The RUNX family has been linked to osteosarcoma via its regulation of many tumorigenicity-related factors. In the regulatory network of cancers, with numerous upstream signaling pathways and its potential target molecules downstream, RUNX is a vital molecule. Hence, a pressing need exists to understand the precise process underpinning the occurrence and prognosis of several malignant tumors. Until recently, RUNX has been regarded as one of the therapeutic targets for bone cancer. Therefore, in this review, we have provided insights into various molecular mechanisms behind the tumorigenic role of RUNX in various important cancers. RUNX is anticipated to grow into a novel therapeutic target with the in-depth study of RUNX family-related regulatory processes, aid in the creation of new medications, and enhance clinical efficacy.

Co-Culture of Mesenchymal Stem Cells and Ligamentocytes on Triphasic Embroidered Poly(L-lactide-co-ε-caprolactone) and Polylactic Acid Scaffolds for Anterior Cruciate Ligament Enthesis Tissue Engineering

Successful anterior cruciate ligament (ACL) reconstructions strive for a firm bone-ligament integration. With the aim to establish an enthesis-like construct, embroidered functionalized scaffolds were colonized with spheroids of osteogenically differentiated human mesenchymal stem cells (hMSCs) and lapine (l) ACL fibroblasts in this study. These triphasic poly(L-lactide-co-ε-caprolactone) and polylactic acid (P(LA-CL)/PLA) scaffolds with a bone-, a fibrocartilage transition- and a ligament zone were colonized with spheroids directly after assembly (DC) or with 14-day pre-cultured lACL fibroblast and 14-day osteogenically differentiated hMSCs spheroids (=longer pre-cultivation, LC). The scaffolds with co-cultures were cultured for 14 days. Cell vitality, DNA and sulfated glycosaminoglycan (sGAG) contents were determined. The relative gene expressions of collagen types I and X, Mohawk, Tenascin C and runt-related protein (RUNX) 2 were analyzed. Compared to the lACL spheroids, those with hMSCs adhered more rapidly. Vimentin and collagen type I immunoreactivity were mainly detected in the hMSCs colonizing the bone zone. The DNA content was higher in the DC than in LC whereas the sGAG content was higher in LC. The gene expression of ECM components and transcription factors depended on cell type and pre-culturing condition. Zonal colonization of triphasic scaffolds using spheroids is possible, offering a novel approach for enthesis tissue engineering.

MiR-27b attenuates dexamethasone-inhibited proliferation and osteoblastic differentiation in MC3T3-E1 cells by targeting PPARγ2.

Osteoporosis is a metabolic bone illness characterized by low bone density and a high risk of fracture. It is estimated that there are >60 million individuals in China suffering from this disease, which highlights an urgent requirement for the development of novel and safe drugs for the long-term treatment of osteoporosis. MicroRNAs (miRNAs/miRs) have previously been identified as critical regulators in the progression of osteoporosis. As an intronic miRNA, miR-27b enhances the osteoblastic differentiation of stem cells from the bone marrow and the maxillary sinus membrane. However, the mechanism underlying miR-27b in osteoporosis remains to be elucidated. In the present study, MC3T3-E1 pre-osteoblasts were treated with dexamethasone (DEX) to establish an in vitro model of osteoporosis. The results of the present study demonstrated that DEX treatment markedly inhibited the viability of MC3T3-E1 cells, and downregulated the expression level of miR-27b. The results of reverse transcription-quantitative PCR, western blotting and dual-luciferase assays revealed that miR-27b directly regulated and suppressed the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) in MC3T3-E1 cells. Furthermore, overexpression of miR-27b by transfection of cells with miR-27b mimic attenuated DEX-mediated inhibition of cell viability, alkaline phosphatase (ALP) activity and the expression levels of bone morphogenetic protein-2 (BMP2), runt-related protein 2 (Runx2) and osteocalcin (OCN). The results of the present study indicated that miR-27b alleviated DEX-inhibited proliferation and osteoblastic differentiation. Moreover, miR-27b knockdown repressed MC3T3-E1 cell viability, ALP activity and protein levels of BMP2, Runx2 and OCN. However, these effects were abrogated by small interfering RNA-mediated PPARγ2 silencing. In conclusion, the results of the present study demonstrated that miR-27b attenuated DEX-inhibited proliferation and osteoblastic differentiation in MC3T3-E1 pre-osteoblasts by targeting PPARγ2.

Zfhx4 regulates endochondral ossification as the transcriptional platform of Osterix in mice

Endochondral ossification is regulated by transcription factors that include SRY-box transcription factor 9, runt-related protein 2 (Runx2), and Osterix. However, the sequential and harmonious regulation of the multiple steps of endochondral ossification is unclear. This study identified zinc finger homeodomain 4 (Zfhx4) as a crucial transcriptional partner of Osterix. We found that Zfhx4 was highly expressed in cartilage and that Zfhx4 deficient mice had reduced expression of matrix metallopeptidase 13 and inhibited calcification of cartilage matrices. These phenotypes were very similar to impaired chondrogenesis in Osterix deficient mice. Coimmunoprecipitation and immunofluorescence indicated a physical interaction between Zfhx4 and Osterix. Notably, Zfhx4 and Osterix double mutant mice showed more severe phenotype than Zfhx4 deficient mice. Additionally, Zfhx4 interacted with Runx2 that functions upstream of Osterix. Our findings suggest that Zfhx4 coordinates the transcriptional network of Osterix and, consequently, endochondral ossification.

Fluid Flow Mechanical Stimulation-Assisted Cartridge Device for the Osteogenic Differentiation of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) have the potential to differentiate into different types of mesodermal tissues. In vitro proliferation and differentiation of hMSCs are necessary for bone regeneration in tissue engineering. The present study aimed to design and develop a fluid flow mechanically-assisted cartridge device to enhance the osteogenic differentiation of hMSCs. We used the fluorescence-activated cell-sorting method to analyze the multipotent properties of hMSCs and found that the cultured cells retained their stemness potential. We also evaluated the cell viabilities of the cultured cells via water-soluble tetrazolium salt 1 (WST-1) assay under different rates of flow (0.035, 0.21, and 0.35 mL/min) and static conditions and found that the cell growth rate was approximately 12% higher in the 0.035 mL/min flow condition than the other conditions. Moreover, the cultured cells were healthy and adhered properly to the culture substrate. Enhanced mineralization and alkaline phosphatase activity were also observed under different perfusion conditions compared to the static conditions, indicating that the applied conditions play important roles in the proliferation and differentiation of hMSCs. Furthermore, we determined the expression levels of osteogenesis-related genes, including the runt-related protein 2 (Runx2), collagen type I (Col1), osteopontin (OPN), and osteocalcin (OCN), under various perfusion vis-à-vis static conditions and found that they were significantly affected by the applied conditions. Furthermore, the fluorescence intensities of OCN and OPN osteogenic gene markers were found to be enhanced in the 0.035 mL/min flow condition compared to the control, indicating that it was a suitable condition for osteogenic differentiation. Taken together, the findings of this study reveal that the developed cartridge device promotes the proliferation and differentiation of hMSCs and can potentially be used in the field of tissue engineering.

RUNX2 as a promising therapeutic target for malignant tumors.

The transcription factor runt-related protein 2 (RUNX2) has an important impact on the transformation of bone marrow mesenchymal stem cells to osteoblasts. Further studies have shown that RUNX2 plays a key role in the invasion and metastasis of cancers. RUNX2 is a “key” molecule in the regulatory network comprised of multiple signaling pathways upstream and its target downstream molecules. Due to the complex regulatory mechanisms of RUNX2, the specific mechanism underlying the occurrence, development and prognosis of malignant tumors has not been fully understood. Currently, RUNX2 as a promising therapeutic target for cancers has become a research hotspot. Herein, we reviewed the current literature on the modulatory functions and mechanisms of RUNX2 in the development of malignant tumors, aiming to explore its potential clinical application in the diagnosis, prognosis and treatment of tumors.

The Effects of Hierarchical Micro/Nano-Structured Titanium Surface on Osteoblast Proliferation and Differentiation Under Diabetic Conditions.

The aim of the present study was to mimic the hierarchical structure of bone tissues by simple sandblasting/acid-etching and anodization to investigate the effects of such surface characteristics on proliferation and differentiation of osteoblasts in high glucose concentrations. By the way, the effects of high glucose levels on osteoblast functions were tested. MC3T3-E1 cells cultured on sand-blasted and acid-etched (SLA) surface and nano-modified SLA (NMSLA) surface were subjected to normal serum (NS) and diabetic serum (DS), respectively. The surface characteristics were evaluated by scanning electron microscopy. Cell proliferation was assessed using MTT assay. The levels of alkaline phosphatase (ALP) activity and mineralization were measured and compared. Real-time polymerase chain reaction was applied to detect the expression levels of osteogenic genes. NMSLA significantly increased cell proliferation at time points ranging from 3 to 7 days under both serums. Cells cultured on NMSLA surfaces displayed significantly higher ALP activities and mineralization. The expression levels of Runx2 (indicates runt-related protein 2), collagen I (COL1), and osteocalcin (OCN) were notably increased on NMSLA surface compared with SLA surface. Moreover, we found that high glucose increased osteoblast proliferation but decreased differentiation of osteoblast slightly. The hierarchical micro/nano-structured titanium surface has a favorable biocompatibility on simultaneously improving osteoblast proliferation and differentiation in diabetic serum.

Enterococcus faecalis attenuates osteogenesis through activation of p38 and ERK1/2 pathways in MC3T3‐E1 cells

To explore the role of Enterococcus faecalis in the proliferation, apoptosis and differentiation of osteoblasts. Pre-osteoblastic MC3T3-E1 cells were treated with heat-killed E.faecalis ATCC 29212 and clinical E.faecalis P25RC strains, respectively. Cell proliferation, mineralized calcium deposition, alkaline phosphatase (ALP) activity and apoptosis were assessed at various time-points. The expression levels of osteogenic-related genes including ALP, osteocalcin (OC), runt-related protein 2 (Runx2) and collagen type 1 (COL1) were also analysed throughout the duration of the experiment. Additionally, the involvement of mitogen-activated protein kinases (MAPKs) signalling pathways was analysed by Western blotting. In the presence of culture supernatant from E.faecalis-treated murine macrophages, apoptosis of MC3T3-E1 cells was detected with flow cytometry. Data were analysed using analysis of variance (anova), and P<0.05 was considered significantly different. E.faecalis significantly inhibited proliferation (P<0.05) and also significantly induced apoptosis of MC3T3-E1 cells (P<0.05), whilst differentiation seemed to be unaffected after 7days of E.faecalis treatment. However, osteogenic differentiation was significantly inhibited with 21-day E.faecalis treatment (P<0.05). The p38 and ERK1/2 phosphorylation pathways associated with mineral deposition and apoptosis were significantly activated in MC3T3-E1 cells. The culture supernatants from E.faecalis-treated macrophages induced osteoblast apoptosis. E.faecalis exerted an inhibitory effect on osteogenesis in pre-osteoblastic MC3T3-E1 cells via phosphorylation of p38 and ERK1/2.

Notoginsenoside R1 stimulates osteogenic function in primary osteoblasts via estrogen receptor signaling

Notoginsenoside R1 (NGR1), a novel phytoestrogen isolated from Panax notoginseng, has been widely used in the treatment of microcirculatory diseases in Asian countries. Here we investigated the effect of NGR1 on osteoblast differentiation and mineralization process. Furthermore, we also evaluated NGR1's estrogenic properties, especially its effects on estrogen receptors (ERs). NGR1 activated the transcriptional activity of phosphorylated estrogen response element (pERE)-luciferase (Luc) and induced ERα phosphorylation in hBMSC. In addition, ER activation correlated with induction and was associated with osteoblast differentiation biomarkers including alkaline phosphatase activity and transcription of osteoblastic genes, e.g., type I collagen (COL1), osteonectin, osteocalcin (OC), runt related protein 2 (Runx2), and osterix. NGR1 also promoted the mineralization process of osteoblasts. The NGR1-induced effects were confirmed to be mediated by the ER by the observation that pretreatment of the osteoblasts with the ER antagonist, ICI 182,780 fully blocked the effects. Our results showed that NGR1 stimulates osteogenic differentiation of cultured osteoblasts by activating ER signaling and in turn might be a potential therapeutic alternative for the prevention and treatment of osteoporosis.

Runx1 is required for hematopoietic defects and leukemogenesis in Cbfb-MYH11 knock-in mice.

CBFβ-SMMHC, the fusion protein generated by the chromosome 16 inversion fusion gene, CBFB-MYH11, is known to initiate leukemogenesis. However, the mechanism through which CBFβ-SMMHC contributes to leukemia development is not well understood. Previously it was proposed that CBFβ-SMMHC acts by dominantly repressing the transcription factor RUNX1, but we recently showed that CBFβ-SMMHC has activities that are independent of RUNX1 repression. In addition, we showed that a modified CBFβ-SMMHC with decreased RUNX1 binding activity accelerates leukemogenesis. These results raise questions about the importance of RUNX1 in leukemogenesis by CBFβ-SMMHC. To test this, we generated mice expressing Cbfb-MYH11 in a Runx1 deficient background, resulting from either homozygous Runx1 null alleles (Runx1−/−) or a single dominant negative Runx1 allele (Runx1+/lz). We found that loss of Runx1 activity rescued the differentiation defects induced by Cbfb-MYH11 during primitive hematopoiesis. During definitive hematopoiesis, RUNX1 loss also significantly reduced the proliferation and differentiation defects induced by Cbfb-MYH11. Importantly, Cbfb-MYH11 induced leukemia had much longer latency in Runx1+/lz mice than in Runx1 sufficient mice. These data indicate that Runx1 activity is critical for Cbfb-MYH11 induced hematopoietic defects and leukemogenesis.

Effects of a hydroxyapatite-coated nanotube surface of titanium on MC3T3-E1 cells: an in vitro study.

To compare the biological behavior of mouse osteoblast-like cells (MC3T3-E1) on hydroxyapatite (HA)-coated nanotube surface of titanium and plasma-sprayed HA (HA-PS)-coated titanium surface. The HA-coated nanotube surface of titanium were fabricated by anodization coupled with alternative immersion method (AIM). MC3T3-E1 osteoblast cells cultured in vitro were seeded onto these different surfaces; their growth states were examined by a confocal laser scanning microscope; the proliferation behavior, alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and analysis of osteoblastic gene expressions were also compared in detail. Significant increases in ALP activity and OCN production on days 7 and 14 (P < 0.05) were observed for AIM-coated HA (HA-AIM) surfaces. However, cells cultured on HA-AIM-coated surfaces showed a delayed growth pattern. Real-time polymerase chain reaction analyses showed significantly higher relative mRNA expression levels of osteoblastic genes (runt-related protein 2, osterix, osteopontin, OCN) in cells cultured on the HA-AIM-coated nanotube surfaces as compared with cells cultured on the HA-PS and baer Ti surfaces. The current research showed that the HA-AIM-coated nanotubular Ti surfaces enhance osteoblast differentiation, which had the potential to further improve osseointegration.

Oleanolic acid exerts an osteoprotective effect in ovariectomy-induced osteoporotic rats and stimulates the osteoblastic differentiation of bone mesenchymal stem cells in vitro

Oleanolic acid (OA) and its glycosides have been reported to prevent bone loss by inhibiting the formation of osteoclasts. However, because bone formation and resorption are balanced processes in bone metabolism, no studies have described the effect of OA on osteogenesis. The aim of the present study was to evaluate the osteoprotective effect of OA in rats with ovariectomy (OVX)-induced osteoporosis and to search for the molecular targets of OA in bone mesenchymal stem cells (bMSCs). Two-month-old female mice that underwent OVX were treated with OA (20 mg/kg a day). After 2 weeks and after 3 months, bone mass was evaluated by micro-CT, morphometry, and immunohistochemical detection. In addition, the expression of 256 genes was measured via microarray and confirmed by real-time reverse transcription-polymerase chain reaction. The effects of OA on the activities of bMSCs were also observed in vitro using alkaline phosphatase and cell proliferation assays. Micro-CT displayed only a tendency for bone loss at 2 weeks but a decrease in bone mass at 3 months after OVX. OA treatment increased osteoblast number, increasing osteocalcin and runt-related protein 2 protein levels in vivo and facilitating the osteoblastic differentiation of bMSCs in vitro at doses of 10(-6) and 10(-5) M. Gene expression profile analysis revealed that OVX caused a marked dysregulation of gene expression, especially at 2 weeks, some of which was rescued by OA. Few of these genes overlapped, but their functions were involved in the Notch signaling pathway between two phases of the osteoporotic process. OA exerts an osteoprotective effect in OVX-induced osteoporotic rats and stimulates the osteoblastic differentiation of bMSCs in vitro. The molecular mechanism of this effect might be related to the Notch signaling pathway and requires further investigation.

RUNX2 expression in developing human bones and various bone tumors

The heterozygous germline mutation of runt-related protein 2 (RUNX2) causes cleidocranial dysplasia. To clarify the involvement of RUNX2 in human osteogenesis, fetal bones and various bone tumors were immunohistochemically examined. During both membranous and endochondral ossification in the fetus (n= 8), RUNX2 was expressed not only in osteoblastic cells but also in surrounding mesenchymal cells and early stage chondrocytes. Such an expression pattern was recapitulated in bone tumors: RUNX2 was unequivocally expressed in osteosarcoma (n= 20) and fibrous dysplasia (n= 10), regardless of the site of occurrence, cell morphology or amount of neoplastic osteoid. RUNX2 expression was limited to less differentiated cells in chondrogenic tumors (n= 20). We further analyzed whether RUNX2 expression was regulated by bone morphogenetic protein-2 (BMP-2), which is critical for osteoblastic differentiation. With real-time polymerase chain reaction, the RUNX2 mRNA level was correlated with BMP-2 mRNA level, and both levels were significantly higher in three osteosarcoma cell lines than in three chondrosarcoma cell lines. With treatment of recombinant BMP-2, the RUNX2 mRNA level was significantly altered in these cell lines. RUNX2 expression is constitutive in developing and neoplastic human osteogenesis, and is most likely to be regulated by BMP-2.

Synergistic Inhibition of Endochondral Bone Formation by Silencing Hif1α and Runx2 in Trauma-induced Heterotopic Ossification

Angiogenesis and osteogenesis are tightly coupled during bone development. We studied the effect of inhibition of Hif1α and Runt-related protein 2 (Runx2) on the formation of heterotopic ossification (HO). We constructed lentivirus vectors expressing Hif1α small interfering RNA (siRNA) and Runx2 siRNA. The inhibition of Hif1α function impaired osteoblast proliferation while osteoblasts differentiated normally. Osteoblasts lacking Runx2 proliferated normally while the differentiation was impaired. The osteoblast differentiation was significantly inhibited by co-Runx2 and Hif1α siRNA treatment. The formation of HO by inhibiting Runx2 and Hif1α in an animal model induced by Achilles tenotomy was investigated. The results showed that lacking of Runx2 and Hif1α could inhibit HO formation. Inhibition of Hif1α prevented HO formation only at the initial step and inhibition of Runx2 worked both at the initial step and after chondrogenesis. Angiogenesis and the expressions of osteogenic genes were downregulated in the Hif1α siRNA group. We found synergistic inhibition of endochondral bone formation by silencing Hif1α and Runx2. Our study provided new insight into the roles of Hif1α and Runx2 during the processes of endochondral bone formation, and had important implications for the new therapeutic methods to inhibit HO or to enhance bone formation.

Effects of enamel matrix proteins on proliferation, differentiation and attachment of human alveolar osteoblasts

Enamel matrix proteins (EMPs) have been demonstrated to promote periodontal regeneration. However, effects of EMPs on human alveolar osteoblasts (hAOBs), up to now, have still been unclear. The purpose of this study was to investigate influence of EMPs on proliferation, differentiation and attachment of hAOBs in vitro. EMPs were extracted using the acetic acid method, hAOBs were obtained and cultured in vitro. Cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of osteogenic markers and cell attachment were measured in the absence and in the presence of EMPs (50, 100 and 200 μg/ml). EMPs increased proliferation of hAOBs; however, they inhibited ALP activity and mRNA expression of osteogenic markers (collagen I, ALP, runt-related protein 2, osteocalcin, bone sialoprotein and osteopontin). Meanwhile, EMPs hindered hAOBs' attachment. These effects occurred in EMPs concentration-dependent manner. These results indicate that EMPs may inhibit osteoblastic differentiation and attachment to prevent ankylosis and allow other cell types to regenerate periodontal tissues.

Osteogenic potential of mesenchymal cells embedded in the provisional matrix after a 6‐week healing period in augmented and non‐augmented extraction sockets: an immunohistochemical prospective pilot study in humans

The aim of the present clinical study was the evaluation of the osteogenic potential of mesenchymal cells embedded in the provisional matrix of non-augmented and with Bio-Oss collagen-augmented human extraction sockets after 6 weeks of healing time. Twenty-five patients with 47 extraction sites participated in the present study. After tooth removal, the extraction sockets were augmented with Bio-Oss collagen or not augmented. At implant placement, bone biopsies of the extraction sockets were obtained. The immunohistochemical analysis of the osteogenic potential of the mesenchymal cells in the provisional matrix was performed using three monoclonal antibodies: core-binding factor α1 (Cbfa1)/runt-related protein (Runx)2, osteonectin (OSN/secreted protein acidic and rich in cyst [SPARC]) and osteocalcin (OC). The statistical analysis was performed using two-factorial analysis for repeated measures, Mann-Whitney U-test and Spearman's rank-order correlation coefficient. Of 47 extraction sockets examined, 17 sockets demonstrated an almost complete ossification. Hence, the provisional matrix of the 30 remaining extraction sockets (21 non-augmented, 9 augmented) was immunohistochemically investigated. No evidence of acute or chronic inflammation was noted in any of the specimens. In the provisional matrix of the non-grafted socket, the median amount of Cbfa1/Runx2-positive cells was 72.3%, of OSN (SPARC) 66.9% and of OC 23.4%, whereas in the grafted sockets the median rate of immunopositive cells staining with Cbfa1/Runx2 was 73.3%, of OSN (SPARC) 61.4% and of OC 20.1%. There was no significant difference in the proportion of positive cells expressed by Cbfa1/Runx2, OSN/SPARC and OC between the grafted and non-grafted socket. Furthermore, the cell density did not correlate to the quantity of stained cells independent of the used proteins. After a 6-week healing period, the provisional matrix was demonstrated to have a high proportion of cells displaying a maturation of mature osteoprogenitor cells to osteoblasts. The grafting procedure did not influence the quantity of osteogenic cells in the extraction socket.

Non-virus-mediated transfer of siRNAs against Runx2 and Smad4 inhibit heterotopic ossification in rats

Heterotopic ossification of muscles, tendons and ligaments are a common problem affecting patient with trauma or received elective surgery. But the existing preventive or therapeutic methods all have disadvantages. Runt-related protein 2 (Runx2) and Smad4 are two regulators that have important roles in the differentiation of osteoblast. In this study, we attempted to examine the effect of Runx2 and Smad4 on the development of heterotopic ossification in vitro. We constructed non-virus-containing small interference RNAs (siRNAs) against Runx2 and Smad4 and tested it with reverse transcriptase-PCR and western blot. We then analyzed the independent effect of Runx2- and Smad4-specific siRNAs and their cooperative effect on the formation of heterotopic ossification induced by trauma in rats. The effects were measured with computed tomography scanning, hematoxylin and eosin staining and immunohistochemistry. We found that the Runx2- and Smad4-specific siRNAs inhibited the expression of Runx2 and Smad4 at the level of messenger RNA and protein. Runx2 and Smad4 independently inhibited the formation of heterotopic ossification. Moreover, their co-expression significantly enhanced the inhibition of heterotopic ossification compared with the independent effect. We suggest that gene therapy to inhibit Runx2 and Smad4 by RNAi could be a powerful approach to prevent or treat heterotopic ossification.

The Downregulation of Onzin Expression and Its Potential Role through Physical and Functional Interaction with PU.1 in AML Cell Differentiation.

Abstract 1006Poster Board I-28Onzin is a small, novel and highly conserved cysteine-rich protein with unique structure and tissue-restricted expression, at the high levels in myeloid, intestine and spleen as well as at lower level in lung. Previous reports indicate that inhibition of endogenous onzin results in a reduced growth rate and an increased sensitivity to apoptotic stimuli, while the enforced expression of onzin in fibroblasts leads to marked apoptotic resistance, loss of G2/M checkpoint control and tumorigenic conversion. However, both the regulation of its expression and its biological role remain greatly elusive. In this study, we provided the first demonstration that onzin expression is significantly downregulated during differentiation induction of acute myeloid leukemic (AML) cell lines and primary cells from AML patients by phorbol 12-myristate 13-acetate (PMA) and all-trans retinoic acid (ATRA). Further, this suppression of onzin expression can also be found in PMA-treated solid tumor cell lines with physiological onzin expression by screening 9 different tumor cells. To explore the potential mechanism of onzin downregulation, next we used PMA as the probe in the following experiment. Applying chemical selective inhibitors and transfected expression of dominant negative mutants or constitutive catalytic form of the related kinases, we showed that Protein kinase C-epsilon (PKCε)-extracellular signal-regulated protein kinase (ERK) signaling axis is required for PMA-induced downregulation of onzin expression. Then, to investigate whether the downregulation of onzin expression is associated with leukemic cell differentiation, the full-length onzin cDNA together with empty vector as a control were respectively transfected into NB4 cells as well as U937 cells by retrovirus infection. NB4 and U937 cells with onzin infection expressed higher level of onzin protein could sufficiently overcome the decrease of endogenous onzin protein induced by PMA. Compared with NB4 and U937 cells with empty vector infection, interestingly, onzin overexpression inhibited PMA- or ATRA-induced cell differentiation, evidenced by CD11b and CD14 expression, NBT assays and morphologic alterations. Finally, to exploit how onzin affects PMA- and ATRA-induced differentiation, we investigated whether onzin interacts with the Runt-related protein 1(Runx1), CCAAT/enhancer-binding protein α (C/EBPα) and PU.1, three important transcriptional factors in hematological cell differentiation. CO-IP results indicate that onzin can effectively interact with PU.1 in 293T and U937 cells, in which the two proteins are respectively ectopically expressed and endogenously expressed, but onzin failed to interact with Runx1 or C/EBPα. Furthermore, we examined the effect of onzin on pM-CSFR-luc (driven by human macrophage colony-stimulating factor receptor promoter containing Runx1, C/EBPα and PU.1 binding sites) activity when transiently transfected with Runx1/CBFα, C/EBPα or PU.1 and onzin alone or together in 293T cells. The results showed that although onzin transefected alone had no effect on M-CSFR-luc activity, onzin can partially but significantly decreased M-CSFR-luc activity driven by PU.1, while the forced expression of onzin also had no effect on the M-CSFR-luc activity driven by Runx1 and C/EBPα. Taken together, this is the first report demonstrating that PMA and ATRA downregulates onzin expression through PKCε-ERK signaling pathway, which favors leukemic cell differentiation. Disclosures:No relevant conflicts of interest to declare.

Stimulation of osteoblasts with Emdogain increases the expression of specific mineralization markers

The purpose of this study was to determine the effects of enamel matrix derivative on mRNA expression of markers related to periodontal healing. Murine osteoprogenitor cells (MC3T3-E1) were grown for 12 and 16 days in mineralization media and stimulated with 100 microg/mL Emdogain (EMD). Cell cultures treated with 2% and 10% fetal calf serum (FCS) served as control. The mRNA expression of bone sialoprotein (BSP), osteopontin (OPN), and runt-related protein 2 (Runx2) was analyzed by real-time polymerase chain reaction. One-way analysis of variance was used for statistical analysis. Stimulation with EMD significantly (P < .01) enhanced mRNA expression of BSP up to 13.9-fold and of OPN up to 3.2-fold at day 16 compared with the 2% FCS control. The expression of mRNA for transcription factor Runx2 was not significantly changed. The beneficial effects seen in periodontal regeneration after treatment with EMD may be related to an increase of the mineralization markers BSP and OPN at mRNA level.