Aire Disruption Influences the Medullary Thymic Epithelial Cell Transcriptome and Interaction With Thymocytes.

The function of medullary thymic epithelial cells (mTECs) is associated with thymocyte adhesion, which is crucial for the negative selection of autoreactive thymocytes in the thymus. This process represents the root of central tolerance of self-components and prevents the onset of autoimmune diseases. Since thymic epithelia correspond to an important target of donor T cells during the onset of chronic graft-vs-host-disease, mTEC-thymocyte adhesion may have implications for alloimmunity. The Aire and Fezf2 genes function as transcriptome controllers in mTECs. The central question of this study is whether there is a mutual relationship between mTEC-thymocyte adhesion and the control of the mTEC transcriptome and whether Aire is involved in this process. Here, we show that in vitro mTEC-thymocyte adhesion causes transcriptome changes in mTECs and upregulates the transcriptional expression of Aire and Fezf2, as well as cell adhesion-related genes such as Cd80 or Tcf7, among others. Crispr-Cas9-mediated Aire gene disruption demonstrated that this gene plays a role in the process of mTEC-thymocyte adhesion. Consistent with the nuclear localization signal (NLS) encoded by Aire exon 3, which was targeted, we demonstrate that Aire KO-/- mTECs impair AIRE protein localization in the nucleus. Consequently, the loss of function of Aire reduced the ability of these cells to adhere to thymocytes. Their transcriptomes differed from their wild-type Aire+/+ counterparts, even during thymocyte adhesion. A set of mRNA isoforms that encode proteins involved in cell adhesion were also modulated during this process. This demonstrates that both thymocyte interactions and Aire influence transcriptome profiling of mTEC cells.

Posttranscriptional Interaction Between miR-450a-5p and miR-28-5p and STAT1 mRNA Triggers Osteoblastic Differentiation of Human Mesenchymal Stem Cells.

We demonstrate that the interaction between miR-450a-5p and miR-28-5p and signal transducer and activator of transcription 1 (STAT1) mRNA correlates with the osteoblastic differentiation of mesenchymal stem cells from human exfoliated deciduous teeth (shed cells). STAT1 negatively regulates runx-related transcription factor 2 (RUNX2), which is an essential transcription factor in this process. However, the elements that trigger osteoblastic differentiation and therefore pause the inhibitory effect of STAT1 need investigation. Usually, STAT1 can be posttranscriptionally regulated by miRNAs. To test this, we used an in vitro model system in which shed cells were chemically induced toward osteoblastic differentiation and temporally analyzed, comparing undifferentiated cells with their counterparts in the early (2 days) or late (7 or 21 days) periods of induction. The definition of the entire functional genome expression signature demonstrated that the transcriptional activity of a large set of mRNAs and miRNAs changes during this process. Interestingly, STAT1 and RUNX2 mRNAs feature contrasting expression levels during the course of differentiation. While undifferentiated or early differentiating cells express high levels of STAT1 mRNA, which was gradually downregulated, RUNX2 mRNA was upregulated toward differentiation. The reconstruction of miRNA-mRNA interaction networks allowed the identification of six miRNAs (miR-17-3p, miR-28-5p, miR-29b, miR-29c-5p, miR-145-3p, and miR-450a-5p), and we predicted their respective targets, from which we focused on miR-450a-5p and miR-28-5p STAT1 mRNA interactions, whose intracellular occurrence was validated through the luciferase assay. Transfections of undifferentiated shed cells with miR-450a-5p or miR-28-5p mimics or with miR-450a-5p or miR-28-5p antagonists demonstrated that these miRNAs might play a role as posttranscriptional controllers of STAT1 mRNA during osteoblastic differentiation. J. Cell. Biochem. 118: 4045-4062, 2017. © 2017 Wiley Periodicals, Inc.

Poly(Vinylidene Fluoride-Trifluorethylene)/barium titanate membrane promotes de novo bone formation and may modulate gene expression in osteoporotic rat model.

Osteoporosis is a chronic disease that impairs proper bone remodeling. Guided bone regeneration is a surgical technique that improves bone defect in a particular region through new bone formation, using barrier materials (e.g. membranes) to protect the space adjacent to the bone defect. The polytetrafluorethylene membrane is widely used in guided bone regeneration, however, new membranes are being investigated. The purpose of this study was to evaluate the effect of P(VDFTrFE)/BT [poly(vinylidene fluoride-trifluoroethylene)/barium titanate] membrane on in vivo bone formation. Twenty-three Wistar rats were submitted to bilateral ovariectomy. Five animals were subjected to sham surgery. After 150 days, bone defects were created and filled with P(VDF-TrFE)/BT membrane or PTFE membrane (except for the sham and OVX groups). After 4 weeks, the animals were euthanized and calvaria samples were subjected to histomorphometric and computed microtomography analysis (microCT), besides real time polymerase chain reaction (real time PCR) to evaluate gene expression. The histomorphometric analysis showed that the animals that received the P(VDF-TrFE)/BT membrane presented morphometric parameters similar or even better compared to the animals that received the PTFE membrane. The comparison between groups showed that gene expression of RUNX2, BSP, OPN, OSX and RANKL were lower on P(VDF-TrFE)/BT membrane; the gene expression of ALP, OC, RANK and CTSK were similar and the gene expression of OPG, CALCR and MMP9 were higher when compared to PTFE. The results showed that the P(VDF-TrFE)/BT membrane favors bone formation, and therefore, may be considered a promising biomaterial to support bone repair in a situation of osteoporosis.

Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction.

We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire(+) mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion.

Bosentan, an endothelin receptor antagonist, ameliorates collagen-induced arthritis: the role of TNF-α in the induction of endothelin system genes.

OBJECTIVE: Endothelins (ETs) are involved in several inflammatory events. The present study investigated the efficacy of bosentan, a dual ETA/ETB receptor antagonist, in collagen-induced arthritis (CIA) in mice. TREATMENT: CIA was induced in DBA/1J mice. Arthritic mice were treated with bosentan (100 mg/kg) once a day, starting from the day when arthritis was clinically detectable. METHODS: CIA progression was assessed by measurements of visual clinical score, paw swelling and hypernociception. Histological changes, neutrophil infiltration and pro-inflammatory cytokines were evaluated in the joints. Gene expression in the lymph nodes of arthritic mice was evaluated by microarray technology. PreproET-1 mRNA expression in the lymph nodes of mice and in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time PCR. The differences were evaluated by one-way ANOVA or Student's t test. RESULTS: Oral treatment with bosentan markedly ameliorated the clinical aspects of CIA (visual clinical score, paw swelling and hyperalgesia). Bosentan treatment also reduced joint damage, leukocyte infiltration and pro-inflammatory cytokine levels (IL-1ß, TNFα and IL-17) in the joint tissues. Changes in gene expression in the lymph nodes of arthritic mice returned to the levels of the control mice after bosentan treatment. PreproET mRNA expression increased in PBMCs from rheumatoid arthritis (RA) patients but returned to basal level in PBMCs from patients under anti-TNF therapy. In-vitro treatment of PBMCs with TNFα upregulated ET system genes. CONCLUSION: These findings indicate that ET receptor antagonists, such as bosentan, might be useful in controlling RA. Moreover, it seems that ET mediation of arthritis is triggered by TNFα.

Nanoscale oxidative patterning of metallic surfaces to modulate cell activity and fate.

In the field of regenerative medicine, nanoscale physical cuing is clearly becoming a compelling determinant of cell behavior. Developing effective methods for making nanostructured surfaces with well-defined physicochemical properties is thus mandatory for the rational design of functional biomaterials. Here, we demonstrate the versatility of simple chemical oxidative patterning to create unique nanotopographical surfaces that influence the behavior of various cell types, modulate the expression of key determinants of cell activity, and offer the potential of harnessing the power of stem cells. These findings promise to lead to a new generation of improved metal implants with intelligent surfaces that can control biological response at the site of healing.

Microarray-based gene expression analysis of human osteoblasts in response to different biomaterials.

Several biomaterials have been widely used in bone regeneration/substitution procedures in orthopedic and oral surgery. However, how these biomaterials alter osteoblast gene expression is poorly understood. We therefore attempted to address this question by using cDNA microarray technique to identify genes that are differentially regulated in osteoblasts exposed to biomaterials comprehending the biocompatibility spectrum of bioactive (bioglass and hydroxyapatite), bioinert (Ti and stainless steel), and biotolerant (polymethylmethacrylate). By using a cDNA microarray containing 687 human IMAGE sequences, we identified in primary cultures of osteoblastic cells differentiated from the human bone marrow and exposed to these biomaterials, genes whose expression was significantly upregulated or downregulated. Among the differentially expressed genes we have found those involved with cell cycle regulation, cell differentiation and proliferation, apoptosis, cell adhesion, bone mineralization and skeletal development. These results can be relevant to a better understanding of the molecular mechanism underlying the behavior of osteoblasts in bone regenerative procedures.

The effect of TAK-778 on gene expression of osteoblastic cells is mediated through estrogen receptor.

This study evaluated the effect of TAK-778 [(2R, 4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide)] on in vitro osteogenic events and on gene expression of osteoblastic cells derived from human alveolar bone and the participation of estrogen receptors (ERs) on such effect. Osteoblastic cells were subcultured, with or without TAK-778 (10(-5) M), to evaluate cell growth and viability, total protein content, and alkaline phosphatase (ALP) activity at 7, 14, and 21 days; bone-like formation at 21 days; and gene expression, using cDNA microarray, at 7 days. Also, osteoblastic cells were exposed to TAK-778 (10(-5) M) combined to ICI182,780, a nonspecific ER antagonist (10(-6) M), and gene expression was evaluated by real-time polymerase chain reaction (PCR) at 7 days. TAK-778 induced a reduction in culture growth and an increase in cell synthesis, ALP activity, and bone-like formation. The cDNA microarray showed genes associated with cell adhesion and differentiation, skeletal development, ossification, and transforming growth factor-beta receptor signaling pathway, with a tendency to be higher expressed in cells exposed to TAK-778. The gene expression of ALP, osteocalcin, Msh homeobox 2, receptor activator of NF-kappa B ligand, and intercellular adhesion molecule 1 was increased by TAK-778 as demonstrated by real-time PCR, and this effect was antagonized by ICI182,780. The present results demonstrated that TAK-778 acts at a transcriptional level to enhance the in vitro osteogenic process and that its effect on gene expression of osteoblastic cells is mediated, at least partially, through ERs. Based on these findings, TAK-778 could be considered in the treatment of bone metabolic disorders.