Runx Expression Research Articles

The regenerative potential of Tideglusib and CHIR99021 small molecules as potent odontogenic differentiation enhancers of human dental pulp stem cells.

To assess the effect of Tideglusib and CHIR99021 small molecules on the odontogenic differentiation potential of human dental pulp stem cells (hDPSCs) via Wnt/β-catenin pathway activation. hDPSCs were isolated from impacted third molars indicated for extraction and were characterized by flow cytometry. hDPSCs were then induced to differentiate into odontogenic lineage in the presence of Tideglusib and CHIR99021. Odontogenic differentiation was evaluated using Alizarin Red stain and RT-PCR for expression of odontogenic specific differentiation markers: DSPP, DMP1, ALP, OPN, and RUNX2 in relation to undifferentiated cells. RT-PCR was also conducted to assess the expression of Wnt/β-catenin pathway activation marker (AXIN2). One-way ANOVA Kruskal-Wallis test was used for statistical analysis. Wnt/β-catenin pathway was successfully activated by Tideglusib and CHIR99021 in hDPSCs where AXIN2 was significantly upregulated. Successful odontogenic differentiation was confirmed by Alizarin Red staining of calcified nodules. RT-PCR for odontogenic differentiation markers DSPP, DMP1, and RUNX expression by hDPSCs induced by CHIR99021 was higher than that expressed by hDPSCs induced by Tideglusib, whereas expression of OPN and ALP was higher in Tideglusib-induced cells than in CHIR99021-induced cells. Both small molecules successfully induced odontogenic differentiation of hDPSCs through Wnt/β-catenin pathway activation. These findings suggest that Tideglusib and CHIR99021 can be applied clinically in pulp regeneration to improve strategies for vital pulp regeneration and to promote dentine repair.

Runt-related transcription factors in human carcinogenesis: a friend or foe?

Cancer is one of the deadliest pathologies with more than 19 million new cases and 10 million cancer-related deaths across the globe. Despite development of advanced therapeutic interventions, cancer remains as a fatal pathology due to lack of early prognostic biomarkers,therapy resistanceand requires identification of novel drug targets. Runt-related transcription factors (Runx) familycontrols several cellular and physiological functions includingosteogenesis. Recent literatures from PubMed was mined and the review was written in comprehensive manner RESULTS: Recent literature suggests that aberrant expression of Runx contributes to tumorigenesis of many organs. Conversely, cell- and tissue-specific tumor suppressor roles of Runx are also reported. In this review, we have provided the structural/functional properties of Runx isoforms and its regulation in context of human cancer. Moreover, in an urgent need to discover novel therapeutic interventions against cancer, we comprehensively discussed the reported oncogenic and tumor suppressive roles of Runx isoforms in several tumor types and discussed the discrepancies that may have risen on Runx as a driver of malignant transformation. Runx may be a novel therapeutic target against a battery of deadly human cancers.

Gene Expression in MC3T3-E1 Cells Treated with Diclofenac and Methylprednisolone.

Nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids (GCs) are often used to treat articular-skeletal disorders. The extended use of NSAIDs and GCs have adverse effects on bone metabolism, reducing bone quality and impairing fracture healing. In the present study, we used mouse pre-osteoblast cells MC3T3-E1 to demonstrate the effects of diclofenac (DF) and methylprednisolone (MP) on cell proliferation and gene expression. Cells were incubated with three doses of DF or MP: 0.5 µM, 5 µM, and 50 µM. MP decreased cell viability even after 24 h, but DF inhibited cell viability after only seven days of treatment. The cells were lysed after one, two, three, and seven days of treatment, and gene expression was analyzed by reverse transcription and quantitative PCR (RT-qPCR) assays. DF did not significantly affect the expression of the osteogenic marker genes. MP modified the expression of Osx, Runx, and Col1a1. We concluded that MP is a more potent inhibitor of mouse pre-osteoblast differentiation and viability than is DF. Our results suggest that prolonged DF treatment could be less harmful to osteoblasts than MP treatment.

Bovine serum albumin-functionalized graphene-decorated strontium as a potent complex nanoparticle for bone tissue engineering

Graphene and its family have a great potential in tissue engineering because of their super mechanical properties, electrical conductivity and antibacterial properties. Considering other properties of graphene such as high surface area and ready-to-use functionalization according to the high oxygen-containing groups in graphene oxide family, some needs could be addressed in bone tissue engineering. Herein, we synthesized and decorated strontium nanoparticles (SrNPs) during the reduction process of graphene oxide using green and novel method. Without using hydrazine or chemical linkers, strontium NPs were synthesized and decorated on the surface of rGO simultaneously using BSA. The results of the UV–Vis, FTIR and Raman spectroscopy demonstrated that BSA could successfully reduce graphene oxide and decorated SrNPs on the surface of rGO. FESEM and TEM exhibited that in situ synthesized SrNPs had 25–30 nm diameter. Interestingly, cell viability for MC3T3-E1 cells treated with SrNPs-rGO, were significantly higher than BSA-rGO and GO in constant concentration. Furthermore, we investigated the alkaline phosphatase activity (ALP) of these nanosheets that the results demonstrated Sr-BSA-rGO enhanced ALP activity more than GO and BSA-rGO. Remarkably, the relative expression of RUNX 2 and Col1 genes of MC3T3-E1 cells was boosted when treated with Sr-BSA-rGO nanosheets. This study revealed that using proteins and other biomolecules as green and facile agent for decoration of smart nanoparticles on the surface of nanosheets, would be promising and assist researcher to replace the harsh and toxic hydrazine like materials with bio-friendly method. These results demonstrated that Sr-BSA-rGO had the excellent capability for regenerating bone tissue and could be used as an osteogenesis booster in implants.

Pan-cancer landscape of the RUNX protein family reveals their potential as carcinogenic biomarkers and the mechanisms underlying their action

BackgroundThe RUNX family of transcription factors plays an important regulatory role in tumor development. Although the importance of RUNX in certain cancer types is well known, the pan-cancer landscape remains unclear.Materials and MethodsData from The Cancer Genome Atlas (TCGA) provides a pan-cancer overview of the RUNX genes. Hence, herein, we performed a pan-cancer analysis of abnormal RUNX expression and deciphered the potential regulatory mechanism. Specifically, we used TCGA multi-omics data combined with multiple online tools to analyze transcripts, genetic alterations, DNA methylation, clinical prognoses, miRNA networks, and potential target genes.ResultsRUNX genes are consistently overexpressed in esophageal, gastric, pancreatic, and pan-renal cancers. The total protein expression of RUNX1 in lung adenocarcinoma, kidney renal clear cell carcinoma (KIRC), and uterine corpus endometrial carcinoma (UCEC) is consistent with the mRNA expression results. Moreover, increased phosphorylation on the T14 and T18 residues of RUNX1 may represent potential pathogenic factors. The RUNX genes are significantly associated with survival in pan-renal cancer, brain lower-grade glioma, and uveal melanoma. Meanwhile, various mutations and posttranscriptional changes, including the RUNX1 D96 mutation in invasive breast carcinoma, the co-occurrence of RUNX gene mutations in UCEC, and methylation changes in the RUNX2 promoter in KIRC, may be associated with cancer development. Finally, analysis of epigenetic regulator co-expression, miRNA networks, and target genes revealed the carcinogenicity, abnormal expression, and direct regulation of RUNX genes.ConclusionsWe successfully analyzed the pan-cancer abnormal expression and prognostic value of RUNX genes, thereby providing potential biomarkers for various cancers. Further, mutations revealed via genetic alteration analysis may serve as a basis for personalized patient therapies.

Prognostic value and immune characteristics of RUNX gene family in human cancers: a pan-cancer analysis

Background: Runt-related transcription factors (RUNX) are involved in numerous fundamental biological processes and play crucial parts in tumorigenesis and metastasis both directly and indirectly. However, the pan-cancer evidence of the RUNX gene family is not available.Methods: In this study, we analyzed the potential association between RUNX gene family expression and patient’s prognosis, immune cell infiltration, drug response, and genetic mutation data across different types of tumors using based on The Cancer Genome Atlas, Gene Expression Omnibus, and Oncomine database.Results: The results showed that the expression of the RUNX gene family varied among different cancer types, revealing its heterogeneity in cancers and that expression of RUNX2 was lower than that of RUNX1 and RUNX3 across all cancer types. RUNX gene family gene expression was related to prognosis in several cancers. Furthermore, our study revealed a clear association between RUNX gene family expression and ESTIMATE score, RNA stemness, and DNA stemness scores. Compared with RUNX1 and RUNX2, RUNX3 showed relatively low levels of genetic alterations. RUNX gene family genes had clear associations with immune infiltrate subtypes, and their expression was positively related to immune checkpoint genes and drug sensitivity in most cases. Two immunotherapy cohorts confirm that the expression of RUNX was correlated with the clinical response of immunotherapy.Conclusions: These findings will help to elucidate the potential oncogenic roles of RUNX gene family genes in different types of cancer and it can function as a prognostic marker in various malignant tumors.

Chicago sky blue 6B (CSB6B), an allosteric inhibitor of macrophage migration inhibitory factor (MIF), suppresses osteoclastogenesis and promotes osteogenesis through the inhibition of the NF-κB signaling pathway

Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory mediator involved in various pathophysiological and inflammatory states. Accumulating line of evidence suggests a role for MIF in regulating bone metabolism and therefore a prime candidate for therapeutic targeting. In this study, we showed that Chicago sky blue 6B (CSB6B) suppresses RANKL-induced osteoclast and bone resorption in vitro via the inhibition of NF-κB signaling activation and promoting proteasome-mediated degradation of MIF. Consequently, the induction of NFATc1 was impaired resulting in downregulation of NFATc1-responsive osteoclast genes. We also demonstrated that CSB6B treatment enhanced primary calvarial osteoblast differentiation and bone mineralization in vitro via the suppression of NF-κB activation and upregulation of Runx expression. Using two murine models of osteolytic bone disorders, we further showed that administration of CSB6B protected mice against pathological inflammatoryc calvarial bone destruction induced by titanium particles mice as well as estrogen-deficiency induced bone loss as a result of ovariectomy. Together, as an MIF inhibitor, CSB6B can inhibit osteoclast differentiation and bone resorption function and enhance the mineralization of osteoblasts through the inhibition of NF-κB pathway. MIF is a prime target for therapeutic targeting for the treatment of osteolytic bone disorders and the MIF inhibitor CSB6B could be potential anti-osteoporosis drug.

Effects of labeling human mesenchymal stem cells with superparamagnetic zinc-nickel ferrite nanoparticles on cellular characteristics and adipogenesis/osteogenesis differentiation.

An attractive cell source for stem cell-based therapy are WJ-MSCs. Hence, tracking WJ-MSCs using non-invasive imaging procedures (such as MRI) and contrast agents (Zn0.5Ni0.5Fe2O4, NFNPs) are required to evaluate cell distribution, migration, and differentiation. Results showed that the bare and dextrin-coated NFNPs were internalized inside the WJ-MSCs and had no effect on the cell viability, proliferation, apoptosis, karyotyping, and morphology of WJ-MSCs up to 125µg/mL. Besides, treated WJ-MSCs were differentiated into osteo/adipocyte-like cells. The expression of RUNX 2, SPP 1 (P < 0.05), and OCN (P > 0.05) genes in the WJ-MSCs treated with dextrin-coated NFNPs was higher than the untreated WJ-MSCs; and the expression of CFD, LPL, and PPAR-γ genes was reduced in WJ-MSCs treated with both NFNPs in comparison with the untreated WJ-MSCs (P > 0.05). Overall, results showed that dextrin-coated NFNPs had no adverse effect on the cellular characteristics, proliferation, and differentiation of WJ-MSCs, and suggesting their potential clinical efficacy.

Abstract 2937: The regulation of FGFR signaling by RTK adaptor protein down-regulation through CROX (cluster regulation of RUNX) theory in DNPC

Abstract Up-regulation of the FGFR signaling is frequently-encountered in DNPC: AR-, NE- prostatic cancer and contributes to the maintenance of these cancer cells. Even though, the importance of active FGFR signaling was recognized as a biomarker in DNPC, still core oncogenic driver of DNPC remained unknown. We formerly reported the importance of Pan RUNX (RUNX1,2,3) as an oncogenic driver. Here, we added a new evaluation factors to the examination of CRPC patient cohort to categorize potentially existing DNPC clearly. Pan RUNX was up-regulated in DNPC patients. The inhibition of Pan RUNX reduced the proliferation of DNPC cells significantly, suggesting that Pan RUNX is functionally and critically involved in the cell proliferation mechanism of DNPC. The inhibition of Pan RUNX inhibited FGFR signaling dramatically. On the other hand, the inhibition of FGFR decreased the transactivation of Pan RUNX. To resolve this contradiction, we identified candidate genes which are mediating the crosstalk between FGFR signaling and Pan RUNX. In addition to our formerly reported S100A16, we isolated and examined the adaptor protein family responsible for RTK activation, which are transcriptionally regulated by the RUNX family. In addition, from the analysis by using the CRPC patient cohorts, Pan RUNX expression and SOS1 etc. genes levels were significantly higher in DNPC rather than those of the other subtypes. RUNX family regulates the FGFR signaling pathway in DNPC cells through transactivating expression, rendering itself an ideal target in anti-tumor strategy toward this cancer. Mechanistically, ChIP-qPCR assay and luciferase reporter experiments further validated RUNX family interacts with the RUNX binding DNA sequence located in the candidate genes promoter and positively regulates it. Knockdown of Pan RUNX led to the decreased expression of the adaptor genes as well as dephosphorylation of FGFR, subsequently suppressed the proliferation of DNPC cells. These data suggest that RUNX depletion-mediated growth inhibitory effect on DNPC cells depends on FGFR signal inactivation via transcriptional regulation of adaptor protein. To achieve cluster regulations of RUNX(CROX), We screened our library and isolated our original Pan RUNX inhibitor based on gene switch technology; Chlorambucil-conjugated Pyrrole Imidazole Polyamide (Chb-M'), which specifically binds to the common RUNX binding consensus sequence(TGTGGT) and inhibits RUNX target genes including the adaptor protein, consistently led to the deactivation of the FGFR signaling pathway and was effective against DNPC cell lines and was exceptionally well-tolerated in mice and exerted excellent efficacy against xenograft mice models of DNPC. Taken together, our work identified a novel interaction of RUNX family and the FGFR signaling pathway in DNPC, which can potentially be exploited in the management of this malignancy. Citation Format: Asami Sasaski, Youhei Yanagida, Hiroshi Sugiyama, Souichi Adachhi, Yasuhiko Kamikubo. The regulation of FGFR signaling by RTK adaptor protein down-regulation through CROX (cluster regulation of RUNX) theory in DNPC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2937.

The Cd8 locus Controls the Functional Conversion of CD4 Th cells to CD4 CTL in the Intestine

Abstract CD4 T cells adapt to their surroundings by differentiating into various T helper (Th) subsets. At steady state, regulatory CD4 T cells (Treg) and interleukin (IL)-17 producing CD4 T cells (Th17 cells) are present in the intestine and localize mainly to the lamina propria, whereas CD4 cytotoxic T lymphocytes (CTL) reside in the epithelium. A hallmark of CD4 CTL is the reactivation of the Cd8 locus and the re-expression of CD8a controlled by the Cd8 enhancer I (E8I). We showed before that CD4 Th cells can reprogram to CTL. Here, we elucidated a mechanism that drives the CTL reprogramming of CD4 Th cells. We identified a long noncoding RNA transcribed from the Cd8 locus, as the critical regulator of the reciprocal expression of T-BET and RUNX combined with the suppression of Foxp3 and Rorc in CD4 CTL precursors. We further found that the E8I region functions as is central coordinator of the RUNX3-controlled cytotoxic gene expression program marked by the re-expression of CD8a. Adoptive transfer of naïve E8I deletion mutant CD4 T cells to Rag1−/− recipient mice led to accelerated weight loss, and severe small and large intestine inflammation with excessive accumulation of pathogenic IL-17A and IL-17A/IFNg double producing CD4 T cells. Altogether, these findings indicate that the Cd8 locus controls the functional polarization of CD4 Th cells and furthermore, that the conversion to CTL not only enhances the direct protective capacity but also regulate the immune response and prevents excessive inflammation and immune pathology at the mucosal border of the intestine.

Mechanical Property of Hydrogels and the Presence of Adipose Stem Cells in Tumor Stroma Affect Spheroid Formation in the 3D Osteosarcoma Model.

Osteosarcoma is one of the most common metastatic bone cancers, which results in significant morbidity and mortality. Unfolding of effectual therapeutic strategies against osteosarcoma is impeded because of the absence of adequate animal models, which can truly recapitulate disease biology of humans. Tissue engineering provides an opportunity to develop physiologically relevant, reproducible, and tunable in vitro platforms to investigate the interactions of osteosarcoma cells with its microenvironment. Adipose-derived stem cells (ASCs) are detected adjacent to osteosarcoma masses and are considered to have protumor effects. Hence, the present study focuses on investigating the role of reactive ASCs in formation of spheroids of osteosarcoma cells (Saos 2) within a three-dimensional (3D) niche, which is created using gellan gum (GG)-silk fibroin. By modifying the blending ratio of GG-silk, the optimum stiffness of the resultant hydrogels such as GG and GG75: S25 is obtained for cancer spheroid formation. This work indicates that the co-existence of cancer and stem cells can form a spheroid, the hallmark of cancer, only in particular microenvironment stiffness. The incorporation of fibrillar silk fibroin within the hydrophilic network of GG in GG75: S25 spongy-like hydrogels closely mimics the stiffness of commercially established cancer biomaterials (e.g., Matrigel, HyStem). The GG75: S25 hydrogel maintains the metabolically active construct for a longer time with elevated expression of osteopontin, osteocalcin, RUNX 2, and bone sialoprotein genes, the biomarkers of osteosarcoma, compared to GG. The GG75: S25 construct also exhibits intense alkaline phosphatase expression in immunohistochemistry compared to GG, indicating itspotentiality to serve as biomimetic niche to model osteosarcoma. Taken together, the GG-silk fibroin-blended spongy-like hydrogel is envisioned as an alternative low-cost platform for 3D cancer modeling.

The Emerging Roles of RUNX Transcription Factors in Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is an evolutionary conserved morphogenetic program necessary for the shaping of the body plan during development. It is guided precisely by growth factor signaling and a dedicated network of specialised transcription factors. These are supported by other transcription factor families serving auxiliary functions during EMT, beyond their general roles as effectors of major signaling pathways. EMT transiently induces in epithelial cells mesenchymal properties, such as the loss of cell-cell adhesion and a gain in cell motility. Together, these newly acquired properties enable their migration to distant sites where they eventually give rise to adult epithelia. However, it is now recognized that EMT contributes to the pathogenesis of several human diseases, notably in tissue fibrosis and cancer metastasis. The RUNX family of transcription factors are important players in cell fate determination during development, where their spatio-temporal expression often overlaps with the occurrence of EMT. Furthermore, the dysregulation of RUNX expression and functions are increasingly linked to the aberrant induction of EMT in cancer. The present chapter reviews the current knowledge of this emerging field and the common themes of RUNX involvement during EMT, with the intention of fostering future research.

A microRNA/Runx1/Runx2 network regulates prostate tumor progression from onset to adenocarcinoma in TRAMP mice.

While decades of research have identified molecular pathways inducing and promoting stages of prostate cancer malignancy, studies addressing dynamic changes of cancer-related regulatory factors in a prostate tumor progression model are limited. Using the TRAMP mouse model of human prostate cancer, we address mechanisms of deregulation for the cancer-associated transcription factors, Runx1 and Runx2 by identifying microRNAs with reciprocal expression changes at six time points during 33 weeks of tumorigenesis. We molecularly define transition stages from PIN lesions to hyperplasia/neoplasia and progression to adenocarcinoma by temporal changes in expression of human prostate cancer markers, including the androgen receptor and tumor suppressors, Nkx3.1 and PTEN. Concomitant activation of PTEN, AR, and Runx factors occurs at early stages. At late stages, PTEN and AR are downregulated, while Runx1 and Runx2 remain elevated. Loss of Runx-targeting microRNAs, miR-23b-5p, miR-139-5p, miR-205-5p, miR-221-3p, miR-375-3p, miR-382-5p, and miR-384-5p, contribute to aberrant Runx expression in prostate tumors. Our studies reveal a Runx/miRNA interaction axis centered on PTEN-PI3K-AKT signaling. This regulatory network translates to mechanistic understanding of prostate tumorigenesis that can be developed for diagnosis and directed therapy.

The Effect of the Combination of Vitamin K2 and Genistein, Coumestrol and Daidzein on the Osteoblast Differentiation and Bone Matrix Formation

Multiple studies have been reported the stimulatory effect of the combinations of nutrients factors on bone formation. One such factor is vitamin K2 which can be associated with bone protective activities. The effect of vitamin K2 alone and in combination with genistein, coumestrol and daidzein on osteoblast differentiation and mineralization were tested. Significantly, vitamin K2 increased bone mineralization in combination with genistein (10-5M), coumestrol (10-7M) and daidzein (10-5M). However, there is no additive effect of this vitamin on alkaline phosphatase (ALP) levels in osteoblasts. By contrast, vitamin K2 enhanced the stimulatory effect of type I collagen and osteocalcin expression. Vitamin K2 alone increased RUNX and OSX expression while there is no synergistic effect with tested compound; this vitamin also did not modulate nuclear factor kappa B ligand (RANKL)/ osteoprotegerin (OPG) ratio expression. These results suggested that vitamin K2 can be more effective factor in the presence of phytoestrogens on the improvement of bone formation after menopause.

Osteoporosis-associated alteration in the signalling status of BMP-2 in human MSCs under adipogenic conditions.

Postmenopausal osteoporosis is characterized by decreased bone quality and mineral density. Mesenchymal stem cells (MSCs) found in the bone marrow, are pluripotent cells able to differentiate into several phenotypes, including osteoblasts and adipocytes. In osteoporosis, MSCs' commitment and differentiation into osteoblast/adipocyte is unbalanced, favoring adipocyte formation. The osteo and adipogenic processes are modulated by the bone morphogenetic protein-2 (BMP-2). This cytokine regulates the expression of transcription factors PPARγ and Runx 2, but its action on cells under adipogenic conditions is poorly understood. In this work we studied BMP-2 signaling in MSCs obtained from bone marrow of control or osteoporotic volunteer postmenopausal women. MSCs were cultured under basal, adipogenic (AD) or AD plus BMP-2 conditions. The protein content of PPARγ, p-PPARγ, Runx2, bone morphogenetic receptor IA (BMPR IA), phosphorylated Smad-1/5/8 (p-Smad) and Smad 4 were determined by specific western blots. mRNA level for BMPRs was determined by PCR and cell localization of p-Smad-1/5/8 were detected by immunocytochemistry. Control MSCs showed a differential response to both AD and AD plus BMP-2 treatments: BMP-2 exerted an anti-adipogenic effect increasing both transcription factors analyzed. Moreover, p-Smads-1/5/8 were detected in nuclei after short term BMP-2 treatment. Osteoporotic MSCs showed no response to exogenous added BMP-2, as shown by p-PPARγ/PPARγ ratio and Runx2 levels, although BMPR-IA level was significantly higher in osteoporotic than in control MSCs. In addition, staining for p-Smad-1/5/8 in o-MSCs was observed around nuclei at all experimental conditions. Taken together results demonstrate failure of BMP-2 signaling in osteoporotic MSCs.

Biomarkers of bone development and oxidative stress in gilthead sea bream larvae fed microdiets with several levels of polar lipids and α-tocopherol

Although dietary marine phospholipids are able to improve culture performance of marine fish larvae in a further extend than soybean lecithin, both types of phospholipids (PL) markedly increase oxidative risk. The inclusion of a fat-soluble antioxidant such as the vitamin E α-tocopherol could allow a better control of oxidative stress. The objective of this study was to determine the combined effect of graded levels of α-tocopherol with different levels and sources of krill phospholipids (KPL) and soybean lecithin (SBL) on growth, survival, resistance to stress, oxidative status, bone metabolism-related genes expression and biochemical composition of sea bream larvae. Sea bream larvae were completely weaned at 16 dph and fed for 30 days seven microdiets with three different levels of PL (0, 40 and 80 g kg−1 diet) and two of α-tocopherol 1500 and 3000 mg kg−1 diet. Sea bream larvae fed diets without PL supplementation showed the lowest survival, growth and stress resistance, whereas increase in PL, particularly KPL, markedly promoted larval survival and growth. However, feeding SBL markedly increased TBARs and GPX gene expression increasing the peroxidation risk in the larvae. Besides, KPL inclusion improved incorporation of n-3 HUFA and, particularly, EPA into larval tissues, these fatty acids being positively correlated with the expression of BMP-4, RUNX 2, ALP, OC and OP genes and to bone mineralization for a given larval size class. The increase in dietary α-tocopherol tends to improve growth in relation to the n-3 HUFA levels in the diet, denoting the protective role of this vitamin against oxidation. Indeed, dietary α-tocopherol decreased the oxidative stress in the larvae as denoted by the reduction in larval TBARs contents and gene expression of SOD and CAT, but not GPX. Thus, increase in dietary α-tocopherol effectively prevented the formation of free radicals from HUFA, particularly EPA, but did not affect the incidence of bone anomalies or the expression of genes related to osteogenetic processes.

Low doses of 15d-PGJ2 induce osteoblast activity in a PPAR-gamma independent manner

Peroxisome proliferator-activated receptor-gamma (PPARγ) regulates both glucose metabolism and bone mass. Evidence suggests that the therapeutic modulation of PPARγ with synthetic agonists activity may elicit undesirable effects on bone. However, there is no information regarding its natural agonist 15d-PGJ2, besides its excellent anti-inflammatory action. In the present study the effects of 15d-PGJ2 on osteoblastic cells were determined. Osteoblastic cells (MC3T3) were cultured in an osteogenic medium in the presence of 1, 3 or 10 μM of 15d-PGJ2 during 21 days and alizarin and Von Kossa staining were employed. The protein expression (type-I collagen, osteonectin, osteopontin, RANKL, osteoprotegerin, HDAC-9c and PPAR-γ) was evaluated after 3 days in the presence of 15d-PGJ2 by western blotting and indirect immunofluorescence methods. The production of mineralized extracellular matrix was observed by transmission electron microscopy. After 72 h of culture, the mRNA was extracted for RT-qPCR analysis of RUNX expression. In the presence of all 3 tested 15d-PGJ2 doses, alizarin red and Von kossa staining were positive demonstrating the ability to the osteoblast differentiation. Type-I collagen and osteonectin proteins expression were up-regulated (p < 0.05) after 72 h in the presence of the smaller doses of 15d-PGJ2. In contrast, osteopontin, RANKL and OPG expression did not significantly alter. In the presence of 15d-PGJ2 it was possible to visualize mineralized nodules in the extracellular matrix confirmed with the increased RUNX mRNA expression. 15d-PGJ2 at small doses increased the osteoblast activity and the bone-related proteins expression.

Interleukin-7 receptor controls development and maturation of late stages of thymocyte subpopulations

Interleukin (IL)-7 is a cytokine essential for T lymphocyte development and homeostasis. However, little is known about the roles of IL-7 receptor α-chain (IL-7Rα) in late stages of T-cell development. To address this question, we established IL-7Rα-floxed mice and crossed them with CD4-Cre transgenic mice. Resultant IL-7R conditional knockout (IL-7RcKO) mice exhibited marked reduction in CD8 single positive (SP) T cells, regulatory T cells (Tregs), and natural killer T (NKT) cells in thymus. The proportion and proliferation of both mature CD4SP and CD8SP thymocytes were decreased without affecting Runx expression. In addition, expression of the glucocorticoid-induced TNF receptor was reduced in CD4SP and CD8SP thymocytes, and expression of CD5 was decreased in CD8SP thymocytes. IL-7RcKO mice also showed impaired Treg and NKT cell proliferation and inhibition of NKT cell maturation. Bcl-2 expression was reduced in CD4SP and CD8SP thymocytes but not in Tregs and NKT cells, and introduction of a Bcl-2 transgene rescued frequency and CD5 expression of CD8SP thymocytes. Furthermore, IL-7RcKO mice exhibited greatly increased numbers of B cells and, to a lesser extent, γδ T and dendritic cells in thymus. Overall, this study demonstrates that IL-7Rα differentially controls development and maturation of thymocyte subpopulations in late developmental stages and suggests that IL-7R expression on αβ T cells suppresses development of other cell lineages in thymus.

RUNX transcription factors: association with pediatric asthma and modulated by maternal smoking.

Intrauterine smoke exposure (IUS) is a strong risk factor for development of airways responsiveness and asthma in childhood. Runt-related transcription factors (RUNX1-3) have critical roles in immune system development and function. We hypothesized that genetic variations in RUNX1 would be associated with airway responsiveness in asthmatic children and that this association would be modified by IUS. Family-based association testing analysis in the Childhood Asthma Management Program genome-wide genotype data showed that 17 of 100 RUNX1 single-nucleotide polymorphisms (SNPs) were significantly (P < 0.03-0.04) associated with methacholine responsiveness. The association between methacholine responsiveness and one of the SNPs was significantly modified by a history of IUS exposure. Quantitative PCR analysis of immature human lung tissue with and without IUS suggested that IUS increased RUNX1 expression at the pseudoglandular stage of lung development. We examined these associations by subjecting murine neonatal lung tissue with and without IUS to quantitative PCR (N = 4-14 per group). Our murine model showed that IUS decreased RUNX expression at postnatal days (P)3 and P5 (P < 0.05). We conclude that 1) SNPs in RUNX1 are associated with airway responsiveness in asthmatic children and these associations are modified by IUS exposure, 2) IUS tended to increase the expression of RUNX1 in early human development, and 3) a murine IUS model showed that the effects of developmental cigarette smoke exposure persisted for at least 2 wk after birth. We speculate that IUS exposure-altered expression of RUNX transcription factors increases the risk of asthma in children with IUS exposure.

Cross-Talk Between Aryl Hydrocarbon and Epidermal Growth Factor Receptors in Human Colon Cancer Cells

G A A b st ra ct s Glut4 (Slc2a4) and the apical glucose transporter Sglt2 (Slc5a2) imply an important role for the stomach in obesity regulation. (c) General: Taurine and glutathione pathway gene expression suggests protective antioxidant properties. Specific expression of various transcription factors, including Barx1, Runx 1-3, and Gata6 as well as neurogenin 3 and Foxa2 emphasize the hetero-cellular nature of this region of the gastrointestinal tract and its differentiation factors. Conclusions: We are able to identify novel unexplored aspects of gastric mucosal function by using subtractive hybridization of RNA from gastric corpus mucosa compared to other regions of the gastrointestinal tract providing a road map for further experimentation.