The essential role of Mkx in periodontal ligament on the metabolism of alveolar bone and cementum.

Introduction: The periodontium is a connective tissue which consists of periodontal ligament, alveolar bone, cementum and gingiva. Periodontal ligament (PDL) is a specialized connective tissue that connects the cementum - coating the surface of the tooth - to the alveolar bone. Mohawk homeobox (Mkx) is a transcription factor that is expressed in PDL, that is known to play a vital role in the development and homeostasis of PDL. A detailed functional analysis of Mkx in the periodontal ligament for alveolar bone and cementum metabolism has not yet been conducted. Materials and methods: Alveolar bone height, bone mineral density (BMD) and bone volume fractions (Bone volume/Total volume: BV/TV) were measured and analyzed using micro-computed tomography (Micro-CT) and 3DBon on 7-week-old male wild-type (WT) (Mkx+/+) (n = 10) and Mkx-knockout (Mkx-/-) (n = 6) rats. Hematoxylin and Eosin (H&E), tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP) and Masson Trichrome staining were performed on 5, 6, and 7-week-old Mkx+/+ and Mkx-/- rats. Cementum surface area and the number of TRAP-positive osteoclasts/mm were quantified, measured, and compared for 5,6 and 7-week-old Mkx+/+ and Mkx-/- rats (n = 3 each). Results: The level of alveolar bone height was significantly higher in Mkx-/- rats than in Mkx+/+ rats. On the other hand, there was significantly less BMD in Mkx-/- alveolar bone. A significant increase in cellular cementum could be observed as early as 5 weeks in Mkx-/- rats when compared with Mkx+/+ rats of the same age. More TRAP-positive osteoclasts were observed in Mkx-/- rats. Conclusion: Our findings further reveal the essential roles of Mkx in the homeostasis of the periodontal tissue. Mkx was found to contribute to bone and cementum metabolism and may be essential to the prevention of diseases such as periodontitis, and could show potential in regenerative treatments.

One-step generation of mice with gene editing by Tol2 transposon-dependent gRNA delivery.

Conditional knockout mice are valuable tools for examining the functions of targeted genes in a time- and space-specific manner. Here, we generated gene-edited mice by using the Tol2 transposon to introduce guide RNA (gRNA) into fertilized eggs obtained by crossing LSL (loxP-stop-loxP)-CRISPR-associated 9 (Cas9) mice, which express Cas9 in a Cre-dependent manner, with CAG-CreER mice. Transposase mRNA and plasmid DNA, which contained a gRNA sequence for the gene encoding tyrosinase flanked by the transposase recognition sequence, were injected together into fertilized eggs. As a result, the transcribed gRNA cleaved the target genome in a Cas9-dependent manner. Using this method, it is possible to generate conditional genome-edited mice more easily in a shorter period of time.

RNA-binding protein LIN28A upregulates transcription factor HIF1α by posttranscriptional regulation via direct binding to UGAU motifs.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates angiogenesis under hypoxic conditions. To investigate the posttranscriptional regulatory mechanism of HIF1α, we performed a cell-based screening to reveal potential cis-elements and the regulatory RNA-binding proteins that act as trans-factors. We found that LIN28A promoted HIF1α protein expression independently of the downregulation of microRNA let-7, which is also directly mediated by LIN28A. Transcriptome analysis and evaluation of RNA stability using RNA-seq and SLAM-seq analyses, respectively, revealed that LIN28A upregulates HIF1A expression via mRNA stabilization. To investigate the physical association of LIN28A with HIF1A mRNA, we performed enhanced crosslinking immunoprecipitation in 293FT cells and integrally analyzed the transcriptome. We observed that LIN28A associates with HIF1A mRNA via its cis-element motif "UGAU". The "UGAU" motifs are recognized by the cold shock domain of LIN28A, and the introduction of a loss-of-function mutation to the cold shock domain diminished the upregulatory activities performed by LIN28A. Finally, the microvessel density assay showed that the expression of LIN28A promoted angiogenesis in vivo. In conclusion, our study elucidated the role of LIN28A in enhancing the HIF1α axis at the posttranscription layer.

The mechanosensitive ion channel PIEZO1 is expressed in tendons and regulates physical performance.

How mechanical stress affects physical performance via tendons is not fully understood. Piezo1 is a mechanosensitive ion channel, and E756del PIEZO1 was recently found as a gain-of-function variant that is common in individuals of African descent. We generated tendon-specific knock-in mice using R2482H Piezo1, a mouse gain-of-function variant, and found that they had higher jumping abilities and faster running speeds than wild-type or muscle-specific knock-in mice. These phenotypes were associated with enhanced tendon anabolism via an increase in tendon-specific transcription factors, Mohawk and Scleraxis, but there was no evidence of changes in muscle. Biomechanical analysis showed that the tendons of R2482H Piezo1 mice were more compliant and stored more elastic energy, consistent with the enhancement of jumping ability. These phenotypes were replicated in mice with tendon-specific R2482H Piezo1 replacement after tendon maturation, indicating that PIEZO1 could be a target for promoting physical performance by enhancing function in mature tendon. The frequency of E756del PIEZO1 was higher in sprinters than in population-matched nonathletic controls in a small Jamaican cohort, suggesting a similar function in humans. Together, this human and mouse genetic and physiological evidence revealed a critical function of tendons in physical performance, which is tightly and robustly regulated by PIEZO1 in tenocytes.

Generation of a tendon-like tissue from human iPS cells.

Tendons and ligaments are essential connective tissues that connect the muscle and bone. Their recovery from injuries is known to be poor, highlighting the crucial need for an effective therapy. A few reports have described the development of artificial ligaments with sufficient strength from human cells. In this study, we successfully generated a tendon-like tissue (bio-tendon) using human induced pluripotent stem cells (iPSCs). We first differentiated human iPSCs into mesenchymal stem cells (iPSC-MSCs) and transfected them with Mohawk (Mkx) to obtain Mkx-iPSC-MSCs, which were applied to a newly designed chamber with a mechanical stretch incubation system. The embedded Mkx-iPSC-MSCs created bio-tendons and exhibited an aligned extracellular matrix structure. Transplantation of the bio-tendons into a mouse Achilles tendon rupture model showed host-derived cell infiltration with improved histological score and biomechanical properties. Taken together, the bio-tendon generated in this study has potential clinical applications for tendon/ligament-related injuries and diseases.

Both microRNA-455-5p and -3p repress hypoxia-inducible factor-2α expression and coordinately regulate cartilage homeostasis.

Osteoarthritis (OA), the most common aging-related joint disease, is caused by an imbalance between extracellular matrix synthesis and degradation. Here, we discover that both strands of microRNA-455 (miR-455), -5p and -3p, are up-regulated by Sox9, an essential transcription factor for cartilage differentiation and function. Both miR-455-5p and -3p are highly expressed in human chondrocytes from normal articular cartilage and in mouse primary chondrocytes. We generate miR-455 knockout mice, and find that cartilage degeneration mimicking OA and elevated expression of cartilage degeneration-related genes are observed at 6-months-old. Using a cell-based miRNA target screening system, we identify hypoxia-inducible factor-2α (HIF-2α), a catabolic factor for cartilage homeostasis, as a direct target of both miR-455-5p and -3p. In addition, overexpression of both miR-455-5p and -3p protect cartilage degeneration in a mouse OA model, demonstrating their potential therapeutic value. Furthermore, knockdown of HIF-2α in 6-month-old miR-455 knockout cartilage rescues the elevated expression of cartilage degeneration-related genes. These data demonstrate that both strands of a miRNA target the same gene to regulate articular cartilage homeostasis.

Mkx regulates the orthodontic tooth movement via osteoclast induction.

INTRODUCTION: The periodontal ligament (PDL) plays an important role in orthodontic tooth movement; however, the underlying molecular mechanism remains unclear. We have previously reported that the Mohawk homeobox (Mkx), a tendon-specific transcription factor, is expressed in the PDL and regulates its homeostasis. MATERIALS AND METHODS: In the present study, we examined the role of Mkx in orthodontic tooth movement via bone remodeling induced by mechanical stimulation in Mkx-deficient rats, which are widely used as experimental animals for orthodontic force application. Orthodontic tooth movement of the maxillary first molar was performed in 7-week-old male Mkx-deficient rats (n = 4) and wild-type Wistar rats (n = 4) using coil springs for 14 days. Hematoxylin and eosin (H&E) staining and tartrate-resistant acid phosphatase (TRAP) staining were performed to evaluate morphological changes and osteoclasts. Furthermore, changes in the expression of receptor activator nuclear factor-kappa B ligand (RANKL) were demonstrated using immunostaining. RESULTS: The amount of tooth movement was significantly lower in Mkx-deficient rats than in wild-type rats. The number of TRAP-positive cells was suppressed in Mkx-deficient rats on the compression side. CONCLUSION: Orthodontic tooth movement experiments in Mkx-deficient rats suggested that Mkx is involved in osteoclast induction at the alveolar bone surface on the compression side. This study reveals the possibility that Mkx plays a mechanosensory role in orthodontic tooth movement by inducing RANKL expression and osteoclastogenesis.

Identification of chemical compounds regulating PD-L1 by introducing HiBiT-tagged cells.

Programmed death-ligand 1 (PD-L1) is a co-inhibitory molecule expressed on tumor cells. Immune checkpoint inhibitors focusing on the PD-L1 mechanism are now being studied for the treatment of various cancer types. However, the regulatory mechanism of PD-L1 is yet to be fully clarified, and a high-throughput system for comparing the abilities of small compounds in regulating PD-L1 has not yet been established. Therefore, we created a HiBiT-tagged lung adenocarcinoma cell line, PC9-KI, for easier and faster detection of changes in PD-L1 protein expression. Using PC9-KI cells, we screened 1280 chemical compounds from the Library of Pharmacologically Active Compounds and identified microtubule polymerization inhibitors and thapsigargin as PD-L1 upregulators and a p97 inhibitor as a PD-L1 downregulator.

MicroRNA Expression Profiling, Target Identification, and Validation in Chondrocytes.

MicroRNAs (miRNAs) are a class of noncoding small RNAs, which play a critical role in various biological processes including musculoskeletal formation and arthritis pathogenesis via regulating target gene expressions, raising the potentially substantial effects on gene expression networks. Over 2000 miRNAs are encoded in the human genome and a single miRNA potentially targets hundreds of genes. To examine the expression and function of miRNAs in chondrocytes and arthritis pathogenesis, we describe the protocols for the current miRNA related experiments including miRNA expression profiling by (1) Next Generation Sequencing and by TaqMan Array system, (2) miRNA target prediction by TargetScan, (3) miRNA target screening by cell-based reporter library assay, and (4) miRNA and its target interaction by HITS-CLIP (high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation) in cartilage and chondrocyte research.

Transcriptome analysis of sevoflurane exposure effects at the different brain regions.

BACKGROUNDS: Sevoflurane is a most frequently used volatile anesthetics, but its molecular mechanisms of action remain unclear. We hypothesized that specific genes play regulatory roles in brain exposed to sevoflurane. Thus, we aimed to evaluate the effects of sevoflurane inhalation and identify potential regulatory genes by RNA-seq analysis. METHODS: Eight-week old mice were exposed to sevoflurane. RNA from medial prefrontal cortex, striatum, hypothalamus, and hippocampus were analysed using RNA-seq. Differently expressed genes were extracted and their gene ontology terms were analysed using Metascape. These our anesthetized mouse data and the transcriptome array data of the cerebral cortex of sleeping mice were compared. Finally, the activities of transcription factors were evaluated using a weighted parametric gene set analysis (wPGSA). JASPAR was used to confirm the existence of binding motifs in the upstream sequences of the differently expressed genes. RESULTS: The gene ontology term enrichment analysis result suggests that sevoflurane inhalation upregulated angiogenesis and downregulated neural differentiation in each region of brain. The comparison with the brains of sleeping mice showed that the gene expression changes were specific to anesthetized mice. Focusing on individual genes, sevoflurane induced Klf4 upregulation in all sampled parts of brain. wPGSA supported the function of KLF4 as a transcription factor, and KLF4-binding motifs were present in many regulatory regions of the differentially expressed genes. CONCLUSIONS: Klf4 was upregulated by sevoflurane inhalation in the mouse brain. The roles of KLF4 might be key to elucidating the mechanisms of sevoflurane induced functional modification in the brain.

The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA.

Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, high-throughput sequencing crosslinking immunoprecipitation (HITS-CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation.

A case of COVID-19 with the atypical CT finding.

COVID-19 usually demonstrates the specific pattern of chest CT findings (GGO, inverted-halo sign, etc). However, some COVID-19 cases show atypical CT findings. Physicians should make comprehensive judgments.

In vitro Neo-Genesis of Tendon/Ligament-Like Tissue by Combination of Mohawk and a Three-Dimensional Cyclic Mechanical Stretch Culture System.

Tendons and ligaments are pivotal connective tissues that tightly connect muscle and bone. In this study, we developed a novel approach to generate tendon/ligament-like tissues with a hierarchical structure, by introducing the tendon/ligament-specific transcription factor Mohawk (MKX) into the mesenchymal stem cell (MSC) line C3H10T1/2 cells, and by applying an improved three-dimensional (3D) cyclic mechanical stretch culture system. In our developed protocol, a combination of stable Mkx expression and cyclic mechanical stretch synergistically affects the structural tendon/ligament-like tissue generation and tendon related gene expression. In a histological analysis of these tendon/ligament-like tissues, an organized extracellular matrix (ECM), containing collagen type III and elastin, was observed. Moreover, we confirmed that Mkx expression and cyclic mechanical stretch, induced the alignment of structural collagen fibril bundles that were deposited in a fibripositor-like manner during the generation of our tendon/ligament-like tissues. Our findings provide new insights for the tendon/ligament biomaterial fields.

Transcriptional, epigenetic and microRNA regulation of growth plate.

Endochondral ossification is a critical event in bone formation, particularly in long shaft bones. Many cellular differentiation processes work in concert to facilitate the generation of cartilage primordium to formation of trabecular structures, all of which occur within the growth plate. Previous studies have revealed that the growth plate is tightly regulated by various transcription factors, epigenetic systems, and microRNAs. Hence, understanding these mechanisms that regulate the growth plate is crucial to furthering the current understanding on skeletal diseases, and in formulating effective treatment strategies. In this review, we focus on describing the function and mechanisms of the transcription factors, epigenetic systems, and microRNAs known to regulate the growth plate.

Post-transcriptional regulation of inflammation by RNA-binding proteins via cis-elements of mRNAs.

In human genome, there are approximately 1,500 RNA-binding proteins (RBPs). They can regulate mRNA stability or translational efficiency via ribosomes and these processes are known as 'post-transcriptional regulation'. Accumulating evidences indicate that post-transcriptional regulation is the determinant of the accurate levels of cytokines mRNAs. While transcriptional regulation of cytokines mRNAs has been well studied and found to be important for the rapid induction of mRNA and regulation of the acute phase of inflammation, post-transcriptional regulation by RBPs is essential for resolving inflammation in the later phase, and their dysfunction may lead to severe autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus. For post-transcriptional regulation, RBPs recognize and directly bind to cis-regulatory elements in 3' untranslated region of mRNAs such as AU-rich or constitutive decay elements and play various roles. In this review, we summarize the recent findings regarding the role of RBPs in the regulation of inflammation.

Ablation of miR-146b in mice causes hematopoietic malignancy.

Excessive and constitutive activation of nuclear factor-κB (NF-κB) leads to abnormal cell proliferation and differentiation, leading to the development of malignant tumors, including lymphoma. MicroRNA 146a (miR-146a) and miR-146b, both of which carry an identical seed sequence, have been shown to contribute to inflammatory diseases and tumors by suppressing the expression of key molecules required for NF-κB activation. However, the functional and physiological differences between miR-146a and miR-146b in disease onset have not been fully elucidated. In this study, we generated miR-146b-knockout (KO) and miR-146a-KO mice by genome editing and found that both strains developed hematopoietic malignancies such as B-cell lymphoma and acute myeloid leukemia during aging. However, the B-cell lymphomas observed in miR-146a- and miR-146b-KO mice were histologically different in their morphology, and the malignancy rate is lower in miR-146b mice than miR-146a mice. Upon mitogenic stimulation, the expression of miR-146a and miR-146b was increased, but miR-146b expression was lower than that of miR-146a. Using a previously developed screening system for microRNA targets, we observed that miR-146a and miR-146b could target the same mRNAs, including TRAF6, and inhibit subsequent NF-κB activity. Consistent with these findings, both miR-146a- and miR-146b-KO B cells showed a high proliferative capacity. Taken together, sustained NF-κB activation in miR-146b KO mice could lead to the development of hematopoietic malignancy with aging.

Pirfenidone may revert the epithelial-to-mesenchymal transition in human lung adenocarcinoma.

The epithelial-to-mesenchymal transition (EMT) in cancer is associated with invasion, metastasis and chemoresistance. Recent studies have revealed the increased expression of programmed death-ligand 1 (PD-L1) in cells undergoing EMT. The underlying mechanism of EMT involves transforming growth factor-ß (TGF-ß) and fibroblast growth factor-2 (FGF-2). Pirfenidone and the known EMT-suppressor nintedanib suppress pulmonary fibrosis partially through suppression of TGF-ß. The present study aimed to determine whether pirfenidone has the potential to induce EMT-reversion, using nintedanib as a reference. The human lung adenocarcinoma cell lines A-549, HCC-827, and PC-9 were treated with TGF-ß and FGF-2 to induce EMT. The EMT-induced cells were further treated with pirfenidone or nintedanib. Phenotypic alterations associated with EMT were assessed by examining the following: i) The expression levels of E-cadherin, vimentin, fibronectin and slug, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescent immunohistochemistry; ii) cell motility via wound-healing assays; and iii) the expression of PD-L1 using RT-qPCR. The combination of TGF-ß and FGF-2 successfully induced EMT in all three cell lines, characterized by a significant reduction in E-cadherin expression in the A-549 and HCC-827 cells, increased expression levels of vimentin, fibronectin, slug and PD-L1, and increased cell motility in all three cell lines. Pirfenidone and nintedanib reverted all of these phenotypes, with the exception of unaltered E-cadherin expression in all three cell lines, and inconsistent expression of vimentin in the HCC-827 and PC-9 cells. Thus, pirfenidone and nintedanib have the ability to induce EMT-reversion in human lung adenocarcinoma.

Potential Activity of Amrubicin as a Salvage Therapy for Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare neuroendocrine carcinoma of the skin with an aggressive clinical course. Although anthracycline- and platinum-based regimens are empirically used as first-line treatments for metastatic or unresectable cases, no salvage therapy has been established. A 73-year-old man with platinum-refractory recurrent MCC was treated with amrubicin. The symptoms improved soon, and a partial response was achieved. A total of nine cycles of amrubicin were administered in nine months with manageable adverse events until disease progression was finally observed. The present findings suggest the potential of amrubicin monotherapy as a second-line therapy for patients with advanced/recurrent MCC.

MYC/BCL2 Double-hit Lymphoma in a Patient with Rheumatoid Arthritis Associated with Methotrexate Treatment.

Several reports have suggested an increased risk of malignant lymphoma in patients with rheumatoid arthritis treated with methotrexate (MTX). We herein describe the case of a 71-year-old woman with rheumatoid arthritis who developed MYC/BCL2 double-hit lymphoma associated with MTX therapy. She developed a fever and lymphadenopathies over a 2-week period and had elevated levels of soluble IL-2 receptor. Inguinal lymph node and bone marrow biopsies showed diffuse large B cell lymphoma. Fluorescent in situ hybridization revealed MYC and BCL2 gene rearrangements in her lymphoma cells. Accordingly, a diagnosis of MYC/BCL2 double-hit lymphoma was made. This is the first reported case of a double-hit lymphoma associated with MTX therapy.