The morbidity and death rates of calcified aortic valves|calcific aortic valve (CAV) disease (CAVD) remain high for its limited therapeutic choices. Here, we investigated the function, therapeutic potential, and putative mechanisms of Enoyl coenzyme A hydratase 1 (ECH1) in CAVD by various in vitro and in vivo experiments. Single-cell sequencing revealed that ECH1 was predominantly expressed in valve interstitial cells and was significantly reduced in CAVs. Overexpression of ECH1 reduced aortic valve calcification in ApoE-/- mice treated with high cholesterol diet, while ECH1 silencing had the reverse effect. We also identified Wnt5a, a noncanonical Wnt ligand, was also altered when ECH1 expression was modulated. Mechanistically, we found that ECH1 exerted anti-calcific actions through suppressing Wnt signaling, since CHIR99021, a Wnt agonist, may significantly lessen the protective impact of ECH1 overexpression on the development of valve calcification. ChIP and luciferase assays all showed that ECH1 overexpression prevented Runx2 binding to its downstream gene promoters (osteopontin and osteocalcin), while CHIR99021 neutralized this protective effect. Collectively, our findings reveal a previously unrecognized mechanism of ECH1-Wnt5a/Ca2+ regulation in CAVD, implying that targeting ECH1 may be a potential therapeutic strategy to prevent CAVD development.

The use of endosseous dental implants may become unfeasible in the presence of significant maxillary bone atrophy; thus, surgical techniques have been proposed to promote bone regeneration in such cases. However, such techniques are complex and may expose the patient to complications. Subperiosteal implants, being placed between the periosteum and the residual alveolar bone, are largely independent of bone thickness. Such devices had been abandoned due to the complexity of positioning and adaptation to the recipient bone site, but are nowadays witnessing an era of revival following the introduction of new acquisition procedures, new materials, and innovative manufacturing methods. We have analyzed the changes induced in gene and protein expression in C-12720 human osteoblasts by differently surface-modified TiO2 materials to verify their ability to promote bone formation. The TiO2 materials tested were (i) raw machined, (ii) electropolished with acid mixture, (iii) sand-blasted + acid-etched, (iv) AlTiColorTM surface, and (v) anodized. All five surfaces efficiently stimulated the expression of markers of osteoblastic differentiation, adhesion, and osteogenesis, such as RUNX2, osteocalcin, osterix, N-cadherin, β-catenin, and osteoprotegerin, while cell viability/proliferation was unaffected. Collectively, our observations document that presently available TiO2 materials are well suited for the manufacturing of modern subperiosteal implants.

Ferroptosis is a new way of cell death, and stimulating the process of cell ferroptosis is a new strategy to treat breast cancer. NGR1 has good anti-cancer activity and is able to slow the progression of breast cancer. However, NGR1 has not been reported in the field related to ferroptosis. By searching the online database for potential targets of NGR1 and the breast cancer disease database, among 11 intersecting genes we focused on Runt-related transcription factor 2 (RUNX2), which is highly expressed in breast cancer, and KEGG pathway enrichment showed that the intersecting genes were mainly enriched in the AGE (advanced glycosylation end products)-RAGE (receptor of AGEs) signaling pathway. After that, we constructed overexpression and down-regulation breast cancer cell lines of RUNX2 in vitro, and tested whether NGR1 treatment induced ferroptosis in breast cancer cells by regulating RUNX2 to inhibit the AGE-RAGE signaling pathway through phenotyping experiments of ferroptosis, Western blot experiments, QPCR experiments, and electron microscopy observation. The results showed that NGR1 was able to inhibit the expression level of RUNX2 and suppress the AGE/PAGE signaling pathway in breast cancer cells. NGR1 was also able to promote the accumulation of Fe2+ and oxidative damage in breast cancer cells by regulating RUNX2 and then down-regulating the expression level of GPX4, FIH1 and up-regulating the expression level of ferroptosis-related proteins such as COX2, ACSL4, PTGS2 and NOX1, which eventually led to the ferroptosis of breast cancer cells.

BACKGROUND: N6-methyladenosine (m6A) methylation is a prevalent RNA modification implicated in various diseases. However, its role in intervertebral disc degeneration (IDD), a common cause of low back pain, remains unclear.
RESULTS: In this investigation, we explored the involvement of m6A demethylation in the pathogenesis of IDD. Our findings revealed that ALKBH5 (alkylated DNA repair protein AlkB homolog 5), an m6A demethylase, exhibited upregulation in degenerative discs upon mild inflammatory stimulation. ALKBH5 facilitated m6A demethylation within the three prime untranslated region (3\'-UTR) of Runx2 mRNA, consequently enhancing its mRNA stability in a YTHDF1 (YTH N6-methyladenosine RNA binding protein F1)-dependent manner. The subsequent elevation in Runx2 expression instigated the upregulation of ADAMTSs and MMPs, pivotal proteases implicated in extracellular matrix (ECM) degradation and IDD progression. In murine models, subcutaneous administration of recombinant Runx2 protein proximal to the lumbar disc in mice elicited complete degradation of intervertebral discs (IVDs). Injection of recombinant MMP1a and ADAMTS10 proteins individually induced mild to moderate degeneration of the IVDs, while co-administration of MMP1a and ADAMTS10 resulted in moderate to severe degeneration. Notably, concurrent injection of the Runx2 inhibitor CADD522 with recombinant Runx2 protein did not result in IVD degeneration in mice. Furthermore, genetic knockout of ALKBH5 and overexpression of YTHDF1 in mice, along with lipopolysaccharide (LPS) treatment to induce inflammation, did not alter the expression of Runx2, MMPs, and ADAMTSs, and no degeneration of the IVDs was observed.
CONCLUSIONS: Our study elucidates the role of ALKBH5-mediated m6A demethylation of Runx2 mRNA in activating MMPs and ADAMTSs, thereby facilitating ECM degradation and promoting the occurrence of IDD. Our findings suggest that targeting the ALKBH5/Runx2/MMPs/ADAMTSs axis may represent a promising therapeutic strategy for preventing IDD.

Histone deacetylase 3 (Hdac3) is an epigenetic regulator of gene expression and interacts with skeletal transcription factors such as Runx2. We previously reported that conditional deletion of Hdac3 in Osterix-Cre recombinase-expressing osteoprogenitor cells (Hdac3 CKOOsx) caused osteopenia and increased marrow adiposity, both hallmarks of skeletal aging. We also showed that Runx2+ cells within osteogenic cultures of Hdac3-depleted bone marrow stromal cells (BMSCs) contain lipid droplets (LDs). Cellular senescence, a non-proliferative metabolically active state, is associated with increased marrow adiposity, bone loss and aging. In this study, we sought to determine if Hdac3 depleted Runx2+ pre-osteoblasts from young mice exhibit chromatin changes associated with early cellular senescence and how these events correlate with the appearance of LDs. We first confirmed that BMSCs from Hdac3 CKOOsx mice have more Runx2 + LD+ cells compared to controls under osteogenic conditions. We then measured senescence-associated distention of satellite DNA (SADS) and telomere-associated foci (TAFs) in Hdac3 CKOOsx and control BMSCs. In situ, Runx2+ cells contained more SADs per nuclei in Hdac3 CKOOsx femora than in controls. Runx2+ BMSCs from Hdac3 CKOOsx mice also contained more SADS and TAFs per nuclei than Runx2+ cells from age-matched control mice in vitro. SADs and TAFs were present at similar levels in Runx2 + LD+ cells and Runx2 + LD- cells from Hdac3 CKOOsx mice. Hdac inhibitors also increased the number of SADS in Runx2 + LD+ and Runx2 + LD- wildtype BMSCs. Senolytics reduced viable cell numbers in Hdac3 CKOOsx BMSC cultures. These data demonstrate that depletion of Hdac3 in osteochondral progenitor cells triggers LD formation and early events in cellular senescence in Runx2+ BMSCs through mutually exclusive mechanisms.
Histone deacetylase 3 (Hdac3) is an enzyme within cells that binds factors in cell nuclei like Runx2 to regulate the expression of genes and control cellular functions. Deleting Hdac3 in cells responsible for bone formation causes bone loss and increases fat in the bone marrow, both hallmarks of skeletal aging. We observed that Hdac3-deletion causes Runx2+ bone marrow stromal cells (BMSCs) to store fats in lipid droplets (LD) even though the cultures were stimulated to become bone cells. Here, we investigated whether these Runx2 + LD+ cells exhibit signs of cellular senescence, which is a zombie-like state associated with increased marrow fat, bone loss and aging. We found that Hdac3-depleted Runx2+ cells showed chromatin changes linked to early cellular senescence alongside the formation of LDs. These findings suggest that Hdac3 plays a crucial role in preventing skeletal aging via regulating both LD formation and cellular senescence in osteochondral progenitor cells.

RUNX2 is a transcription factor crucial for bone formation. Mutant mice with varying levels of Runx2 expression display dosage-dependent skeletal abnormalities, underscoring the importance of Runx2 dosage control in skeletal formation. RUNX2 activity is regulated by several molecular mechanisms, including epigenetic modification such as DNA methylation. In this study, we investigated whether targeted repressive epigenome editing including hypermethylation to the Runx2-DMR/CpG island shore could influence Runx2 expression using Cas9-based epigenome-editing tools. Through the transient introduction of CRISPRoff-v2.1 and gRNAs targeting Runx2-DMR into MC3T3-E1 cells, we successfully induced hypermethylation of the region and concurrently reduced Runx2 expression during osteoblast differentiation. Although the epigenome editing of Runx2-DMR did not impact the expression of RUNX2 downstream target genes, these results indicate a causal relationship between the epigenetic status of the Runx2-DMR and Runx2 transcription. Additionally, we observed that hypermethylation of the Runx2-DMR persisted for at least 24 days under growth conditions but decreased during osteogenic differentiation, highlighting an endogenous DNA demethylation activity targeting the Runx2-DMR during the differentiation process. In summary, our study underscore the usefulness of the epigenome editing technology to evaluate the function of endogenous genetic elements and revealed that the Runx2-DMR methylation is actively regulated during osteoblast differentiation, subsequently could influence Runx2 expression.

Bone mechanotransduction is a critical process during skeletal development in embryogenesis and organogenesis. At the same time, the type and level of mechanical loading regulates bone remodeling throughout the adult life. The aberrant mechanosensing of bone cells has been implicated in the development and progression of bone loss disorders, but also in the bone-specific aspect of other clinical entities, such as the tumorigenesis of solid organs. Novel treatment options have come into sight that exploit the mechanosensitivity of osteoblasts, osteocytes, and chondrocytes to achieve efficient bone regeneration. In this regard, runt-related transcription factor 2 (Runx2) has emerged as a chief skeletal-specific molecule of differentiation, which is prominent to induction by mechanical stimuli. Polycystins represent a family of mechanosensitive proteins that interact with Runx2 in mechano-induced signaling cascades and foster the regulation of alternative effectors of mechanotransuction. In the present narrative review, we employed a PubMed search to extract the literature concerning Runx2, polycystins, and their association from 2000 to March 2024. The keywords stated below were used for the article search. We discuss recent advances regarding the implication of Runx2 and polycystins in bone remodeling and regeneration and elaborate on the targeting strategies that may potentially be applied for the treatment of patients with bone loss diseases.

After spinal cord injury, astrocytes undergo a reactive process and form an astroglial scar, which impedes the regeneration of axons. The role of Runx2 in promoting the transformation of astrocytes in the central nervous system is well-established. However, it remains unclear whether Runx2 also plays a role in the development of astroglial scar, and the precise underlying mechanism has yet to be identified. Recently, our study using cell culture and animal models has demonstrated that Runx2 actually suppresses astrocyte activation and the formation of astroglial scar following injury. The initial results demonstrated an increase in the expression of Runx2 in astrocytes following in vivo injury. Subsequently, the overexpression of Runx2 resulted in the inhibition of astrocyte activation, reduction in the total area of astroglial scar, and restoration of neural function after 14 days of injury. However, these effects were reversed by CADD522. These findings indicate that Runx2 could potentially serve as a therapeutic intervention for spinal cord injury (SCI). Furthermore, our findings suggest that the Nuclear-matrix-targeting signal (NMTS) of Runx2 is associated with its effect. In summary, the study\'s results propose that targeting Runx2 may be a promising treatment approach for reactive astrocytes and astroglial scar in the recovery of SCI.

The osteogenic differentiation process, by which bone marrow mesenchymal stem cells and osteoprogenitors transform into osteoblasts, is regulated by several growth factors, cytokines, and hormones. Plasma Rich in Growth Factors (PRGF) is a blood-derived preparation consisting of a plethora of bioactive molecules, also susceptible to containing epigenetic factors such as ncRNAs and EVs, that stimulates tissue regeneration. The aim of this study was to investigate the effect of the PRGF clot formulation on osteogenic differentiation. Firstly, osteoblast cells were isolated and characterised. The proliferation of bone cells cultured onto PRGF clots or treated with PRGF supernatant was determined. Moreover, the gene expression of Runx2 (ID: 860), SP7 (ID: 121340), and ALPL (ID: 249) was analysed by one-step real-time quantitative polymerase chain reaction (RT-qPCR). Additionally, alkaline phosphatase (ALPL) activity determination was performed. The highest proliferative effect was achieved by the PRGF supernatant in all the study periods analysed. Concerning gene expression, the logRGE of Runx2 increased significantly in osteoblasts cultured with PRGF formulations compared with the control group, while that of SP7 increased significantly in osteoblasts grown on the PRGF clots. On the other hand, despite the fact that the PRGF supernatant induced ALPL up-regulation, significantly higher enzyme activity was detected for the PRGF clots in comparison with the supernatant formulation. According to our results, contact with the PRGF clot could promote a more advanced phase in the osteogenic process, associated to higher levels of ALPL activity. Furthermore, the PRGF clot releasate stimulated a higher proliferation rate in addition to reduced SP7 expression in the cells located at a distant ubication, leading to a less mature osteoblast stage. Thus, the spatial relationship between the PRGF clot and the osteoprogenitors cells could be a factor that influences regenerative outcomes.

In this work, we propose a new technique involving the modification of commercial screen-printed carbon electrodes with electrochemically reduced graphene oxide to serve as the starting point of a future electrochemical biosensor for the detection of two osteogenic biomarkers: alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2). The electrodes were characterized after each modification by cyclic voltammetry and electrochemical impedance spectroscopy, showing the appropriate electrochemical characteristics for each modification type. The results obtained from scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, and contact angle measurements are well correlated with each other, demonstrating the successful modification of the electrodes with graphene oxide and its subsequent reduction. The bioreceptors were immobilized on the electrodes by physical adsorption, which was confirmed by electrochemical methods, structural characterization, and contact angle measurements. Finally, the functionalized electrodes were incubated with the specific target analytes and the detection relied on monitoring the electrochemical changes occurring after the hybridization process. Our results indicated that the pilot platform has the ability to detect the two biomarkers up to 1 nM, with increased sensitivity observed for RUNX2, suggesting that after further optimizations, it has a high potential to be employed as a future biosensor.