Thrap3 promotes osteogenesis by inhibiting the degradation of Runx2.

The dysfunction of osteogenesis is a key character in the pathogenesis of osteoporosis, but the network of signaling mechanisms in controlling the differentiation of osteoblast remain unclear. Thrap3 has been proved participating in various biological process, especially in the differentiation of stem cells. Here, we demonstrate that Thrap3 could promote osteogenesis through the inhibition of the degradation of Runx2, which is a key molecular structure in early osteoblast differentiation. Furthermore, we found that the osteogenesis enhancing capacity of Thrap3 was caused by physically binding with Sox9, inhibiting the transcriptional activity of Sox9, and then decreasing the decomposition-promoted effect of Sox9 on Runx2. Our data shows that Thrap3 promotes osteoblast differentiation through the Thrap3-Sox9-Runx2 axis. What we found may help for further clarifying the molecular mechanism of osteogenic differentiation and give a new potential therapeutic target for osteoporosis.

RUNX1 and RUNX2 transcription factors function in opposing roles to regulate breast cancer stem cells.

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24-/low versus CD24+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFß1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24-/low and CD24+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFß for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.

Runx2 silencing promotes adipogenesis via down-regulation of DLK1 in chondrogenic differentiating MSCs.

BACKGROUND: For cartilage regeneration, stem cells are a promising cell source; however, even the advances made in the differentiation of stem cells into precursor-differentiated cartilage cells have not been successful with respect to reprograming these cells to achieve complete differentiation and fully functioning cells until now. Previous findings suggest that Runx2 plays a major role in chondrocyte differentiation and maturation. Although targeting Runx2 has enhanced some chondrocyte properties, the adipogenic lineage shift has eventually occurred in these cells. The present study mainly aimed to reveal the mechanism of this adipogenesis. METHODS: To create inducible artificial shRNA-miR expressing vectors, the designed short hairpin RNAs (shRNAs) were inserted into the pri-mir-30 backbone, cloned into lentiviral pLVET-Tet-on, and transducted into mesenchymal stem cells (MSCs). Runx2 gene was silenced in MSCs either for 1 week or 4 weeks and cultured in the chondrogenic medium. At days 7, 14 and 28, cells were harvested, and chondrogenesis, adipogenesis and hypertrophic states were examined using histochemical staining and a real-time polymerase chain reaction assay. RESULTS: The results showed that the designed shRNA-miR effectively targeted Runx2 in mRNA and protein levels. Chondrogenic markers were up-regulated in constantly silenced Runx2 group; however, adipogenic markers and fat droplets appeared gradually. DLK1 gene was also significantly down-regulated in this group, and overexpression of DLK1 abrogated adipogenesis in the Runx2 targeted group. CONCLUSIONS: Based on these results, it can be concluded that DLK1 is responsible for the lineage shift in Runx2 targeted chondrogenic differentiating MSCs.

Let-7a-5p inhibits BMSCs osteogenesis in postmenopausal osteoporosis mice.

PURPOSE: The aim of this study was to investigate the mechanism of let-7a-5p in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in postmenopausal osteoporosis (PMOP) mice. METHODS: A mouse model of PMOP was established and osteoporosis model was identified by micro-CT scan. BMSCs in the sham group and PMOP group were cultured and osteogenic differentiation was induced. The expression of let-7a-5p in BMSCs was detected by qRT-PCR, and BMSCs was induced by osteogenic differentiation in sham and PMOP group. The BMSCs treated by let-7a-5p mimics, let-7a-5p inhibitor and negative control were named as let-7a-5p mimics group, mimics NC group, let-7a-5p inhibitor group and inhibitor NC group, respectively. ALP staining and alizarin red staining were used to detect osteogenic differentiation ability, qRT-PCR and western blot were used to detect the expression of Runt-related transcription factor 2 (Runx2) and Osterix. The targeting relationship between let-7a-5p and TGFBR1 were verificated by target scan and luciferase reporter gene assay. RESULTS: The PMOP mouse model was successfully established. The expression of let-7a-5p in BMSCs of PMOP group was significantly higher than that in the sham group (P < 0.05). Let-7a-5p reduced the expression of ALP and the formation of calcified nodules, while also inhibited the expression of Runx2 and Osterix. TGFBR1 is the target gene of let-7a-5p. CONCLUSION: Let-7a-5p might inhibit the osteogenic differentiation of BMSCs in PMOP mice by regulating TGFBR1.

RUNX2 Plays An Oncogenic Role in Esophageal Carcinoma by Activating the PI3K/AKT and ERK Signaling Pathways.

BACKGROUND/AIMS: Esophageal carcinoma is a frequently occurring cancer at upper gastrointestinal tract. We aimed to evaluate the roles and possible mechanism of Runt Related Transcription Factor 2 (RUNX2) in the development of esophageal cancer. METHODS: The expression of RUNX2 in esophageal carcinoma tissues and cells was investigated by qRT-PCR. Effects of RUNX2 on cell viability, apoptosis, migration and invasion were assessed using MTT assay, flow cytometry assay/western blot analysis, and Transwell assays, respectively. Afterwards, effects of RUNX2 on of the activation of the PI3K/AKT and ERK pathways were explored by Western blot analysis. In addition, a PI3K/AKT pathway inhibitor LY294002 and an ERK inhibitor U0126 were applied to further verify the regulatory relationship between RUNX2 and the PI3K/AKT and ERK signaling pathways. Besides, the RUNX2 function on tumor formation in vivo was investigated by tumor xenograft experiment. RESULTS: The result showed that RUNX2 was highly expressed in esophageal carcinoma tissues and cells. Knockdown of RUNX2 significantly inhibited TE-1 and EC-109 cell viability, repressed TE-1 cell migration and invasion, and increased TE-1 cell apoptosis. RUNX2 overexpression showed the opposite effects on HET-1A cells. Moreover, RUNX2-mediated TE-1 cell viability, migration and invasion were associated with the activation of the PI3K/AKT and ERK pathways. Besides, knockdown of RUNX2 markedly suppressed tumor formation in vivo. CONCLUSION: Our results indicate that RUNX2 may play an oncogenic role in esophageal carcinoma by activating the PI3K/ AKT and ERK pathways. RUNX2 may serve as a potent target for the treatment of esophageal carcinoma.

Zaluzanin C (ZC) induces osteoblast differentiation through regulating of osteogenic genes expressions in early stage of differentiation.

Zaluzanin C (ZC) is a sesquiterpene lactones used in herbal medicines. This study examined the effects of ZC on osteoblast differentiation. ZC-induced mRNA expressions levels of osteogenic genes in C3H10T1/2 and MC3T3-E1 cells were determined by RT-PCR and qPCR. ZC regulated the expression of key osteogenic genes in the early stage of differentiation, including distal-less homeobox 5 (Dlx5), DNA-binding protein inhibitor (Id1) and Runt-related transcription factor 2 (Runx2). In addition, ZC increased Runx2 promoter activity, as assessed via a luciferase assay, and Runx2 protein level. These results suggest that ZC may enhance osteoblast differentiation by upregulating the expression of osteogenic genes, especially early stage like as Dlx5, Id1 and Runx2.

Twist1 and Twist2 Contribute to Cytokine Downregulation following Chronic NOD2 Stimulation of Human Macrophages through the Coordinated Regulation of Transcriptional Repressors and Activators.

Proper regulation of microbial-induced cytokines is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, chronic NOD2 stimulation in macrophages decreases cytokines upon pattern recognition receptor (PRR) restimulation; cytokine attenuation to PRR stimulation is similarly observed in intestinal macrophages. The role for the transcriptional repressors Twist1 and Twist2 in regulating PRR-induced cytokine outcomes is poorly understood and has not been reported for NOD2. We found that Twist1 and Twist2 were required for optimal cytokine downregulation during acute and, particularly, chronic NOD2 stimulation of human macrophages. Consistently, Twist1 and Twist2 expression was increased after chronic NOD2 stimulation; this increased expression was IL-10 and TGF-ß dependent. Although Twist1 and Twist2 did not coregulate each other's expression, they cooperated to enhance binding to cytokine promoters after chronic NOD2 stimulation. Moreover, Twist1 and Twist2 contributed to enhance expression and promoter binding of the proinflammatory inhibitor c-Maf and the transcriptional repressor Bmi1. Restoring c-Maf and Bmi1 expression in Twist-deficient macrophages restored NOD2-induced cytokine downregulation. Furthermore, with chronic NOD2 stimulation, Twist1 and Twist2 contributed to the decreased expression and cytokine promoter binding of the transcriptional activators activating transcription factor 4, C/EBPα, Runx1, and Runx2. Knockdown of these transcriptional activators in Twist-deficient macrophages restored cytokine downregulation after chronic NOD2 stimulation. Finally, NOD2 synergized with additional PRRs to increase Twist1 and Twist2 expression and Twist-dependent pathways. Therefore, after chronic NOD2 stimulation Twist1 and Twist2 coordinate the regulation of both transcriptional activators and repressors, thereby mediating optimal cytokine downregulation.

Fenofibrate decreases the bone quality by down regulating Runx2 in high-fat-diet induced Type 2 diabetes mellitus mouse model.

BACKGROUND: This study is to investigate the effect of fenofibrate on the bone quality of Type 2 diabetes mellitus (T2DM) mouse model. METHODS: T2DM mouse model was induced by high-fat-diet, and the mice were treated with fenofibrate (100 mg/kg) (DIO-FENO) or PBS (DIO-PBS) for 4 weeks. The bone microstructure and biomechanical properties of femora were analyzed by micro-CT and 3-Point bending test. The protein expression was detected by immunohistochemical staining and Western blot. The cell apoptosis was evaluated by TUNEL staining. The Bcl2, caspase 3, and osteoblast marker genes were detected by RT-qPCR. RESULTS: The biomechanical properties of bones from DIO-FENO group were significantly lower than those in the control and DIO-PBS groups. Besides, the trabecular number was lower than those of the other groups, though the cortical porosity was decreased compared with that of DIO-PBS group because of the increase of apoptotic cells. The expression of osteocalcin and collagen I were decreased after treatment with fenofibrate in T2DM mice. Moreover, the cell viability was decreased after treated with different concentrations of fenofibrate, and the expression of Runx2 decreased after treated with high dose of fenofibrate. CONCLUSION: Fenofibrate decreases the bone quality of T2DM mice through decreasing the expression of collagen I and osteocalcin, which may be resulted from the down regulation of Runx2 expression.

VIP and CRF reduce ADAMTS expression and function in osteoarthritis synovial fibroblasts.

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family is known to play an important role in the pathogenesis of osteoarthritis (OA), working on aggrecan degradation or altering the integrity of extracellular matrix (ECM). Thus, the main purpose of our study was to define the role of vasoactive intestinal peptide (VIP) and corticotrophin-releasing factor (CRF), as immunoregulatory neuropeptides, on ADAMTS production in synovial fibroblasts (SF) from OA patients and healthy donors (HD). OA- and HD-SF were stimulated with pro-inflammatory mediators and treated with VIP or CRF. Both neuropeptides decreased ADAMTS-4, -5, -7 and -12 expressions, aggrecanase activity, glycosaminoglycans (GAG), and cartilage oligomeric matrix protein (COMP) degradation after stimulation with fibronectin fragments (Fn-fs) in OA-SF. After stimulation with interleukin-1ß, VIP reduced ADAMTS-4 and -5, and both neuropeptides decreased ADAMTS-7 production and COMP degradation. Moreover, VIP and CRF reduced Runx2 and ß-catenin activation in OA-SF. Our data suggest that the role of VIP and CRF on ADAMTS expression and cartilage degradation could be related to the OA pathology since scarce effects were produced in HD-SF. In addition, their effects might be greater when a degradation loop has been established, given that they were higher after stimulation with Fn-fs. Our results point to novel OA therapies based on the use of neuropeptides, since VIP and CRF are able to stop the first critical step, the loss of cartilage aggrecan and the ECM destabilization during joint degradation.

Nitric Oxide Up-Regulates RUNX2 in LNCaP Prostate Tumours: Implications for Tumour Growth In Vitro and In Vivo.

Aberrant expression of the transcription factor RUNX2 in prostate cancer has a number of important consequences including increased resistance to apoptosis, invasion and metastasis to bone. We previously demonstrated that hypoxia up-regulated RUNX2 in tumour cells, which in turn up-regulated the anti-apoptotic factor Bcl-2. Here, we investigate the impact of nitric oxide (NO) on RUNX2 and Bcl-2 expression in prostate cancer and further, how RUNX2 over-expression can impact tumour growth, angiogenesis and oxygenation in vivo. The effect of NO levels on RUNX2 and thus Bcl-2 expression was examined in prostate cancer cells in vitro using methods including gene and protein expression analyses, nitrite quantitation, protein-DNA interaction assays (ChIP) and viability assays (XTT). The effect of RUNX2 over-expression on tumour physiology (growth, oxygenation and angiogenesis) was also assessed in vivo using LNCaP xenografts. A low (but not high) concentration of NO (10 µM) induced expression of RUNX2 and Bcl-2, conferring resistance to docetaxel. These effects were induced via the ERK and PI3K pathways and were dependent on intact AP-1 binding sites in the RUNX2 promoter. RUNX2 over-expression in LNCaP tumours in vivo decreased the time to tumour presentation and increased tumour growth. Moreover, these tumours exhibited improved tumour angiogenesis and oxygenation. Low levels of NO increase expression of RUNX2 and Bcl-2 in LNCaP prostate tumour cells, and in vivo up-regulation of RUNX2 created tumours with a more malignant phenotype. Collectively, our data reveals the importance of NO-regulation of key factors in prostate cancer disease progression.

GnRH regulates trophoblast invasion via RUNX2-mediated MMP2/9 expression.

STUDY HYPOTHESIS: We hypothesized that Runt-related transcription factor 2 (RUNX2), matrix metalloproteinase (MMP)2 and MMP9 are involved in basal and gonadotrophin-releasing hormone (GnRH)-induced human extravillous trophoblast (EVT) cell invasion. STUDY FINDING: Our finding indicates that GnRH-induced RUNX2 expression enhances the invasive capacity of EVT cells by modulating the expression of MMP2 and MMP9. WHAT IS KNOWN ALREADY: GnRH is expressed in first-trimester placenta and exerts pro-invasive effects on EVT cells in vitro. RUNX2 regulates MMP2 and MMP9 expression and is often associated with invasive phenotypes. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: First-trimester human placenta (n = 9) was obtained from women undergoing elective termination of pregnancy. The localization of RUNX2, MMP2 and MMP9 in first-trimester human placenta was examined by immunohistochemistry. Primary or immortalized (HTR-8/SVneo) EVT cells were treated alone or in combination with GnRH, GnRH antagonist Antide, MAPK kinase inhibitor PD98095, phosphatidylinositol 3-kinase inhibitor LY294002, MMP2/9 inhibitor or small interfering RNAs (siRNAs) targeting RUNX2, MMP2 and/or MMP9. Protein and mRNA levels were measured by western blot and RT-PCR, respectively. Cell invasiveness was evaluated by transwell Matrigel or collagen I invasion assays. MAIN RESULTS AND THE ROLE OF CHANCE: RUNX2, MMP2 and MMP9 were detected in the cell column regions of human first-trimester placental villi. GnRH treatment increased RUNX2 mRNA and protein levels in HTR-8/SVneo cells and primary EVTs, and these effects were attenuated by co-treatment with Antide, PD98095 or LY294002. Down-regulation of RUNX2 by siRNA reduced basal and GnRH-induced MMP2/9 expression and cell invasion. Moreover, pharmacological inhibition or siRNA-mediated knockdown of MMP2/9 reduced basal and GnRH-induced cell invasion. LIMITATIONS, REASONS FOR CAUTION: The lack of an in vivo model is the major limitation of our in vitro study. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide important insight into the functions of the GnRH - GnRH receptor system in early implantation and placentation. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This research was supported by Canadian Institutes of Health Research (Grant #143317) to P.C.K.L. The authors have nothing to disclose.

MicroRNA-204 Targets Runx2 to Attenuate BMP-2-induced Osteoblast Differentiation of Human Aortic Valve Interstitial Cells.

Osteoblast differentiation of valve interstitial cells (VICs) is a key step in valve calcification, but the molecular mechanisms involved are not fully understood. In this study, we aimed to investigate whether microRNA (miR)-204-regulated VICs differentiation through modulation of runt-related transcription factor 2 (Runx2), a key transcription factor for osteogenesis. Our data demonstrated that miR-204 was markedly downregulated in both human calcified aortic valves and bone morphogenetic protein (BMP)-2-stimulated aortic VICs. In vitro experiments showed that miR-204 acted as a negative regulator of osteogenic differentiation by repressing Runx2 and thereby inhibiting expression of osteoblast-related genes, including alkaline phosphatase and osteocalcin, which were all induced by BMP-2. Luciferase reporter assays validated Runx2 as the direct target of miR-204. Furthermore, increased alkaline phosphatase activity and osteocalcin expression after miR-204 inhibition were abolished by small interfering RNA-mediated silencing of Runx2. Overall, these data suggested miR-204 as a possible molecular switch inhibiting osteoblastic transdifferentiation of human aortic VICs and targeting miR-204 may have therapeutic potential for human aortic valve calcification.

Effects of rifampicin on osteogenic differentiation and proliferation of human mesenchymal stem cells in the bone marrow.

This study was designed to investigate the effect of different concentrations of rifampicin on osteogenic differentiation and proliferation of mesenchymal stem cells (MSCs) in human bone marrow. Rifampicin treatment at 0, 4, 8, 16, 32, 64, and 128 mg/mL was applied throughout the whole process, from stromal cells purified from human bone marrow to differentiated bone cells. The effect of rifampicin on MSC proliferation was determined using the MTT assay. The effect of rifampicin on the expressions of type I collagen (COL1A1), osteopontin/bone Gla protein (OPN/BGP), and alkaline phosphatase (ALP) in human osteoblast cells were determined by real-time polymerase chain reaction, and the expressions of COL1A1, OPN/BGP, and the runt-related transcription factor (RUNX2) were determined by Western blot. Results showed that the proliferation of MSCs was significantly inhibited when the rifampicin concentration exceeded 32 mg/mL. In addition, increased rifampicin concentrations inhibited the formation of calcium nodules, OPN/BGP, and COL1A1 in osteoblasts after 28 days of induction. The RNA expressions of OPN/BGP, COL1A1, and ALP were significantly downregulated compared to those of the control group in osteoblasts after induction. The protein expressions of RUNX2, COL1A1, and OPN/BGP were also significantly downregulated compared to those of the control group after induction. In conclusion, rifampicin at exorbitant concentration exerts adverse effects on the proliferation of MSCs in human bone marrow and the differentiation of osteoblasts.

Downregulated LncRNA-ANCR promotes osteoblast differentiation by targeting EZH2 and regulating Runx2 expression.

Long noncoding RNAs (lncRNAs) are key regulators of diverse biological processes such as transcriptional regulation, cell growth and differentiation. Previous studies have demonstrated that the lncRNA-ANCR (anti-differentiation ncRNA) is required to maintain the undifferentiated cell state within the epidermis. However, little is known about whether ANCR regulates osteoblast differentiation. In this study, we found that the ANCR expression level is significantly decreased during hFOB1.19 cell differentiation. ANCR-siRNA blocks the expression of endogenous ANCR, resulting in osteoblast differentiation, whereas ANCR overexpression is sufficient to inhibit osteoblast differentiation. We further demonstrated that ANCR is associated with enhancer of zeste homolog 2 (EZH2) and that this association results in the inhibition of both Runx2 expression and subsequent osteoblast differentiation. These data suggest that ANCR is an essential mediator of osteoblast differentiation, thus offering a new target for the development of therapeutic agents to treat bone diseases.

The canonical BMP signaling pathway plays a crucial part in stimulation of dentin sialophosphoprotein expression by BMP-2.

Dentin sialophosphoprotein (DSPP), a typical dentin-specific protein, is mainly expressed in the dentin extracellular matrix and plays a role in dentin mineralization. BMP-2 provides a strong signal for differentiation and mineralization of odontoblasts and osteoblasts. Previously, BMP-2 treatment is reported to stimulate Dspp expression in the MD10-F2 pre-odontoblast cells through activation of the heterotrimeric transcription factor Y (NF-Y). The canonical BMP signaling pathway is known to contribute greatly to biomineralization, however, it is not known whether it is involved in Dspp expression. Here, we investigated this question. Activation of the canonical BMP-2 signaling pathway in MDPC-23, preodontoblast cell, by overexpression of constitutively active Smad1/5 or downstream transcription factors Dlx5 and Runx2 stimulated Dspp expression. Conversely, knockdown of each element with siRNA significantly blocked the BMP-2-induced Dspp expression. To test whether these transcription factors downstream of BMP-2 are directly involved in regulating Dspp, we analyzed the mouse Dspp promoter. There are 5 well conserved homeodomain binding elements, H1 to H5, in Dspp proximal promoter regions (-791 to +54). A serial deletion of H1 and H2 greatly changed basal promoter activity and responsiveness to Dlx5 or Msx2. However, further deletions did not change the responsiveness to Dlx5 or Msx2. H1 and H2 sites can be suggested as specific response elements of Dlx5 and Msx2, respectively, based on their promoter activity modulation. Thus, the canonical BMP-2 signaling pathway plays a crucial part in the regulation of Dspp expression through the action of Smads, Dlx5, Runx2, and Msx2.

The role of runt-related transcription factor 2 (Runx2) in the late stage of odontoblast differentiation and dentin formation.

Runx2, of the Runx family, is an essential transcription factor that controls bone and tooth development by regulating osteoblast and odontoblast differentiation. However, the function of Runx2 in late stage odontoblast differentiation is not clear. We studied the function of Runx2 in dentinogenesis by generating transgenic mice expressing Runx2 specifically in odontoblasts. We observed dentin formation in postnatal day 3 (P3), P7 and P28 mice and measured the expression levels of Runx2 and matrix proteins in dentin. The odontoblasts in transgenic mice (Tg) lost their tall columnar shape and polarization and dentinal tubules were absent. The dental pulp chamber was dramatically enlarged and the dentin in Tg mice was thinner. Osteoblast-like cells were seen instead of normal odontoblasts and were embedded in a bone-like matrix, indicating that dentin formation was replaced with bone. Predentin was disorganized possessing lacunae that contained odontoblasts. The mandibular molars of Tg mice showed noticeable defects by Micro-CT. Using quantitative real-time PCR, the expression of dentin matrix proteins, particularly dentin sialophosphoprotein (DSPP), was found to be upregulated in 3-day-old Tg mice and downregulated at 1 month of age. These findings indicate that Runx2 inhibited odontoblast terminal differentiation and induced transdifferentiation of odontoblasts to osteoblasts at the late cell differentiation stage. Therefore, Runx2 should be inhibited in odontoblasts to encourage normal cell maturation, differentiation and dentinogenesis.

TWIST1 promotes the odontoblast-like differentiation of dental stem cells.

Stem cells derived from the dental pulp of extracted human third molars (DPSCs) have the potential to differentiate into odontoblasts, osteoblasts, adipocytes, and neural cells when provided with the appropriate conditions. To advance the use of DPSCs for dentin regeneration, it is important to replicate the permissive signals that drive terminal events in odontoblast differentiation during tooth development. Such a strategy is likely to restore a dentin matrix that more resembles the tubular nature of primary dentin. Due to the limitations of culture conditions, the use of ex vivo gene therapy to drive the terminal differentiation of mineralizing cells holds considerable promise. In these studies, we asked whether the forced expression of TWIST1 in DPSCs could alter the potential of these cells to differentiate into odontoblast-like cells. Since the partnership between Runx2 and Twist1 proteins is known to control the onset of osteoblast terminal differentiation, we hypothesized that these genes act to control lineage determination of DPSCs. For the first time, our results showed that Twist1 overexpression in DPSCs enhanced the expression of DSPP, a gene that marks odontoblast terminal differentiation. Furthermore, co-transfection assays showed that Twist1 stimulates Dspp promoter activity by antagonizing Runx2 function in 293FT cells. Analysis of our in vitro data, taken together, suggests that lineage specification of DPSCs can be modulated through ex vivo gene modifications.

MiR-373 Inhibits the Epithelial-Mesenchymal Transition of Prostatic Cancer via Targeting Runt-Related Transcription Factor 2.

Prostatic cancer (PCa) is a prevalent form of malignancy based on its high associated levels of mortality and morbidity across the world. MicroRNAs (miRNAs) are significant in the advancement of prostatic cancer. The current study is aimed at exploring the potential roles of miR-373 in PCa. In turn, the study conducted a qRT-PCR test to determine the levels of mRNA. A western blot test was also executed in determining the protein level. The processes of transwell assay and wound healing were integrated in the detection of the potential for PCa cells to invade and migrate. The integration of dual luciferase reporter assay is critical in determining the levels of luciferase activity among prostatic cancer cells. Then, the results showed a net decrease of miR-373 within prostatic cancer cells and tissues. Upregulated miR-373 reduced the invasion and migration potential of PCa cells. Moreover, overexpressed miR-373 increased the levels of E-cadherin and FSP1 as epithelial cell markers. Similarly, the overregulation of miR-373 brought about the upregulation of mesenchymal markers (N-cadherin, Snail, and vimentin). The study predicted runt-related transcription factor 2 (RUNX2) to be a target of miR-373. The luciferase activity of PCa cells was decreased after the cotransfection with miR-373 mimics and RUNX2 3' untranslated region (3'UTR) wild type (WT). Moreover, RUNX2 became upregulated in PCa cells and tissues. The upregulation of miR-373 decreased the mRNA and protein level of RUNX2. However, overexpressed RUNX2 abated the roles of miR-373 in the intrusion and migration of PCa cells and in regulating the expression of epithelial cell markers and mesenchymal markers. In short, miR-373 may regulate the EMT of PCa cells via targeting RUNX2. The miR-373/RUNX2 axis provides a therapeutic target for PCa.

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.

Molecular mechanisms of the inhibitory effect of lipopolysaccharide (LPS) on osteoblast differentiation.

Osteoblasts express Toll like receptor (TLR) 4 and produce osteoclast-activating cytokines in response to the stimulation by lipopolysaccharide (LPS). It has recently been reported that LPS exerts an inhibitory effect on osteoblast differentiation into osteocytes. However, the molecular mechanisms of this inhibitory effect remain ambiguous. The downstream signals of TLR4 are mediated by adaptor molecules including myeloid differentiation factor 88 (MyD88), leading to the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases (ERKs), whose activation by LPS requires the upstream serine/threonine kinase, Cot/Tpl2. To determine the signal molecules responsible for the inhibitory effects of LPS on osteoblast differentiation, we examined the in vitro differentiation of the primary osteoblasts from myd88(-/-) and cot/tpl2(-/-) mice. The matrix mineralization by the wild-type and cot/tpl2(-/-) osteoblasts was significantly inhibited by LPS, whereas that of myd88(-/-) was not affected. During differentiation, LPS suppressed the mRNA expression of runt related transcription factor 2 (Runx2), osterix (Sp7), and activating transcription factor 4 (ATF4) in the wild-type, but not in the myd88(-/-) osteoblasts. The inhibitory effect of LPS on the mRNA expression of these transcription factors was absent in the early phase but partially impaired in the late phase of differentiation in the cot/tpl2(-/-) osteoblasts. Thus, the inhibitory effect of LPS on osteoblast differentiation is Myd88-dependent, whereas the degree of its requirement for Cot/Tpl2 varies depending on the differentiation phase.