Key regulators of vascular calcification in chronic kidney disease: Hyperphosphatemia, BMP2, and RUNX2.

Vascular calcification is quite common in patients with end-stage chronic kidney disease and is a major trigger for cardiovascular complications in these patients. These complications significantly impact the survival rate and long-term prognosis of individuals with chronic kidney disease. Numerous studies have demonstrated that the development of vascular calcification involves various pathophysiological mechanisms, with the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) being of utmost importance. High phosphate levels, bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2) play crucial roles in the osteogenic transdifferentiation process of VSMCs. This article primarily reviews the molecular mechanisms by which high phosphate, BMP2, and RUNX2 regulate vascular calcification secondary to chronic kidney disease, and discusses the complex interactions among these factors and their impact on the progression of vascular calcification. The insights provided here aim to offer new perspectives for future research on the phenotypic switching and osteogenic transdifferentiation of VSMCs, as well as to aid in optimizing clinical treatment strategies for this condition, bearing significant clinical and scientific implications.

Relaxin-2 promotes osteoblastic differentiation mediated by epidermal growth factor and epidermal growth factor receptor signaling.

Loss of osteogenic differentiation potential of osteoblasts has been associated with the pathogenesis of osteoporosis. Thus, stimulation of osteoblastic differentiation is a therapeutic strategy for osteoporosis. Relaxin-2 is a peptide hormone with potent biological functions. However, the effects of Relaxin-2 in osteoblastic differentiation and osteoporosis have not been reported before. Here, we report a novel physiological role of Relaxin-2 in promoting osteoblastic differentiation and mineralization of MC3T3-E1 cells. Our results indicate that exposure to Relaxin-2 upregulated the expression, and elevated the activity of alkaline phosphatase (ALP) when MC3T3-E1 cells were cultured in osteogenic differentiation medium (OM). Additionally, Relaxin-2 upregulated the mRNA levels of osteocalcin (ocn), osteopontin (opn), and collagen type I alpha 1 (Col1a1). The alizarin red S staining assay revealed that Relaxin-2 promoted the mineralization of MC3T3-E1 cells. We also found that Relaxin-2 increased the expression of Runx-2 as well as the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR). Importantly, silencing of EGF abolished the effects of Relaxin-2 in osteoblastic differentiation and related gene expression. These findings suggest that Relaxin-2 stimulates osteogenic differentiation through activating EGF/EGFR signaling.

NFATc1 and RUNX2 Expression on Orthodontic Tooth Movement with Gradually Increasing Force.

Background: Orthodontic tooth movement occurs due to bone resorption and apposition on the pressure and tension side of the PDL. The transcription factors associated with osteoclast differentiation are NFATc1 while osteoblast differentiation is associated with RUNX2. The optimum force of orthodontic tooth movement can move the teeth to the desired position, without causing discomfort and tissue damage to the patient. Objective: This study aims to analyse the effect of gradually increasing force on orthodontic tooth movement (by evaluating the NFATc1 and RUNX2 expression) in rats. Methods: This research is an in vivo experimental study with a post-test control group design. Twenty-eight healthy male adult Wistar rats (Rattus novergicus) aged 4-5 months with body weights 200-250 g rats were divided into seven study groups. Treatment groups in this study are given the force (by applying a closed coil spring between the maxillary central incisor and the maxillary first molar) of 5 g, 5-10 g, 10 g, and 10-20 g with the duration of treatment in 14 and 28 days. After the treatment day was finished, the alveolar bone tissue was isolated and investigated by immunohistochemical methods. Results: Indicate a significant difference between the control and all treatment groups of NFATc1 (p=0.003; p=0.000; p:0.010; p=0.001; p=0.001; p=0.000) and RUNX2 with groups of 10 g/14 days, 10 g/28 days, 5 g/28 days, 10 g/14 days,10-20 g/28 days (p=0.001; p=0.000; p=0.000; p=0.017; p=0.014; p=0.000) values. Conclusion: Gradually increasing force affects orthodontic tooth movement by inducing bone resorption (high expression of NFATc1) in the pressure area and bone apposition (high expression of RUNX2) in the tension area. Applying heavy force by initially applying light force could inhibit hyalinization.

circRUNX2.2, highly expressed in Marek's disease tumor tissues, functions in cis to regulate parental gene RUNX2 expression.

Marek's disease (MD), an immunosuppression disease induced by Marek's disease virus (MDV), is one of the significant diseases affecting the health and productive performance of poultry. The roles of circular RNAs (circRNAs) in MD development were poorly understood. In this study, we found a circRNA derived from exon 6 of RUNX family transcription factor 2 (RUNX2) gene, named circRUNX2.2, was highly expressed in chicken tumorous spleens (TS) induced by MDV. Through fluorescence in situ hybridization and nuclear-cytoplasmic separation assay, we determined circRUNX2.2 was mainly located in the nucleus. Knockout experiments confirmed that the flanking complementary sequences (RCMs) mediated its circularization. Gain of function assay and dual luciferase reporter gene assay revealed that circRUNX2.2 could promote the expression of RUNX2 via binding with its promoter region. RNA antisense purification assay and mass spectrometry assay showed circRUNX2.2 could recruit proteins such as CHD9 protein. Knocking down CHD9 expression decreased the expression of RUNX2 gene, which confirmed the positive regulation that circRUNX2.2 on RUNX2 expression was probably facilitated via recruiting CHD9 protein. Functional experiments showed that circRUNX2.2 promoted the proliferation of the MD lymphoma-derived chicken cell line, MDCC-MSB1, which confirmed the potential oncogenic role of circRNX2.2 in tumor development. In conclusion, we found that the RUNX2-derived circRUNX2.2 can positively regulate the transcription of the parental gene RUNX2 in a cis-acting manner. The high expression of circRUNX2.2 in MD tumor tissues indicated that it might mediate MD lymphoma progression.

RUNX2 regulation in osteoblast differentiation: A possible therapeutic function of the lncRNA and miRNA-mediated network.

Osteogenic differentiation is a crucial process in the formation of the skeleton and the remodeling of bones. It relies on a complex system of signaling pathways and transcription factors, including Runt-related transcription factor 2 (RUNX2). Non-coding RNAs (ncRNAs) control the bone-specific transcription factor RUNX2 through post-transcriptional mechanisms to regulate osteogenic differentiation. The most research has focused on microRNAs (miRNAs) and long ncRNAs (lncRNAs) in studying how they regulate RUNX2 for osteogenesis in both normal and pathological situations. This article provides a concise overview of the recent advancements in understanding the critical roles of lncRNA/miRNA/axes in controlling the expression of RUNX2 during bone formation. The possible application of miRNAs and lncRNAs as therapeutic agents for the treatment of disorders involving the bones and bones itself is also covered.

l-Carnitine relieves cachexia-related skeletal muscle fibrosis by inducing deltex E3 ubiquitin ligase 3L to negatively regulate the Runx2/COL1A1 axis.

BACKGROUND: Cancer cachexia-induced skeletal muscle fibrosis (SMF) impairs muscle regeneration, alters the muscle structure and function, reduces the efficacy of anticancer drugs, diminishes the patient's quality of life and shortens overall survival. RUNX family transcription factor 2 (Runx2), a transcription factor, and collagen type I alpha 1 chain (COL1A1), the principal constituent of SMF, have been linked previously, with Runx2 shown to directly modulate COL1A1 mRNA levels. l-Carnitine, a marker of cancer cachexia, can alleviate fibrosis in liver and kidney models; however, its role in cancer cachexia-associated fibrosis and the involvement of Runx2 in the process remain unexplored. METHODS: Female C57 mice (48 weeks old) were inoculated subcutaneously with MC38 cells to establish a cancer cachexia model. A 5 mg/kg dose of l-carnitine or an equivalent volume of water was administered for 14 days via oral gavage, followed by assessments of muscle function (grip strength) and fibrosis. To elucidate the interplay between the deltex E3 ubiquitin ligase 3L(DTX3L)/Runx2/COL1A1 axis and fibrosis in transforming growth factor beta 1-stimulated NIH/3T3 cells, a suite of molecular techniques, including quantitative real-time PCR, western blot analysis, co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays, were used. The relevance of the DTX3L/Runx2/COL1A1 axis in the gastrocnemius was also explored in the in vivo model. RESULTS: l-Carnitine supplementation reduced cancer cachexia-induced declines in grip strength (>88.2%, P < 0.05) and the collagen fibre area within the gastrocnemius (>57.9%, P < 0.05). At the 5 mg/kg dose, l-carnitine also suppressed COL1A1 and alpha-smooth muscle actin (α-SMA) protein expression, which are markers of SMF and myofibroblasts. Analyses of the TRRUST database indicated that Runx2 regulates both COL1A1 and COL1A2. In vitro, l-carnitine diminished Runx2 protein levels and promoted its ubiquitination. Overexpression of Runx2 abolished the effects of l-carnitine on COL1A1 and α-SMA. Co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays confirmed an interaction between DTX3L and Runx2, with l-carnitine enhancing this interaction to promote Runx2 ubiquitination. l-Carnitine supplementation restored DTX3L levels to those observed under non-cachectic conditions, both in vitro and in vivo. Knockdown of DTX3L abolished the effects of l-carnitine on Runx2, COL1A1 and α-SMA in vitro. The expression of DTX3L was negatively correlated with the levels of Runx2 and COL1A1 in untreated NIH/3T3 cells. CONCLUSIONS: This study revealed a previously unrecognized link between Runx2 and DTX3L in SMF and demonstrated that l-carnitine exerted a significant therapeutic impact on cancer cachexia-associated SMF, potentially through the upregulation of DTX3L.

3D-printed titanium scaffolds loaded with gelatin hydrogel containing strontium-doped silver nanoparticles promote osteoblast differentiation and antibacterial activity for bone tissue engineering.

Bone tissue engineering offers a promising alternative to stimulate the regeneration of damaged tissue, overcoming the limitations of conventional autografts and allografts. Recently, titanium alloy (Ti) implants have garnered significant attention for treating critical-sized bone defects, especially with the advancement of 3D printing technology. Although Ti alloys have impressive versatility, their lack of cellular adhesion, osteogenic and antibacterial properties are significant factors that contribute to their failure. Hence, to overcome these obstacles, this study aimed to incorporate osteoinductive and antibacterial cue-loaded hydrogels into 3D-printed Ti (3D-Ti) scaffolds. 3D-Ti scaffolds were synthesized using the direct metal laser sintering method and loaded with a gelatin (Gel) hydrogel containing strontium-doped silver nanoparticles (Sr-Ag NPs). Compared with Ag NPs, Sr-doped Ag NPs increased the expression of Runx2 mRNA, which is a key bone transcription factor. We subjected the bioactive 3D-hybrid scaffolds (3D-Ti/Gel/Sr-Ag NPs) to physicochemical and material characterization, followed by cytocompatibility and osteogenic evaluation. The microporous and macroporous topographies of the scaffolds with Sr-Ag NPs showed increased Runx2 expression and matrix mineralization, with potent antibacterial properties. Therefore, the 3D-Ti scaffolds incorporated with Sr-Ag NP-loaded Gel hydrogels favored osteoblast differentiation and antibacterial activity, indicating their potential for orthopedic applications.

Long non-coding RNAs in bone formation: Key regulators and therapeutic prospects.

Recent scientific investigations have revealed the intricate mechanisms underlying bone formation, emphasizing the essential role of long non-coding RNAs (lncRNAs) as critical regulators. This process, essential for skeletal strength and functionality, involves the transformation of mesenchymal stem cells into osteoblasts and subsequent deposition of bone matrix. lncRNAs, including HOX transcript antisense RNA (HOTAIR), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), differentiation antagonizing non-coding RNA (DANCR), and maternally expressed gene 3 (MEG3), have emerged as prominent players in this regulatory network. HOTAIR modulates osteoblast differentiation by interacting with chromatin-modifying enzymes, while MALAT1 regulates osteogenic differentiation through microRNA interactions. DANCR collaborates with Runx2 to fine-tune osteoblast differentiation, and MEG3 orchestrates multiple signaling pathways crucial for bone formation. Moreover, other lncRNAs such as H19, lncRNA for enhancing osteogenesis 3, rhabdomyosarcoma 2-associated transcript, urothelial cancer associated 1, taurine up-regulated gene 1, and nuclear enriched abundant transcript 1 contribute to the complex regulatory network governing osteoblast activities. Understanding the precise roles of these lncRNAs offers promising avenues for developing innovative therapeutic strategies targeting bone-related disorders like osteoporosis. Overall, this review summarizes the pivotal role of lncRNAs in bone formation, highlighting their potential as targets for future research endeavors aimed at advancing therapeutic interventions in bone diseases.

Bu-Sui-Dan Enhances Osteoblast Differentiation by Upregulating VGLL4 to Counteract TEAD4-Mediated RUNX2 Transcription Suppression in Ovariectomized Rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Postmenopausal osteoporosis (PMOP) has been considered as a major causative factor for bone-joint pain and inducing pathologic fractures. Bu-Sui-Dan (BSD), a classic ancient herbal formula, has been shown to exhibit osteoprotective effects by promoting bone marrow development and bone growth. However, the exact mechanism of BSD are still unexplored. AIM OF STUDY: The study aimed to investigate the protective effect of BSD against osteoporotic injury, and to explore whether BSD regulated BMSCs' osteogenic differentiation by targeting VGLL4, which in turn improved PMOP. MATERIALS AND METHODS: The anti-osteoporotic effect of BSD was studied in ovariectomized (OVX) rats and bone marrow mesenchymal stem cells (BMSCs). Micro-CT imaging and HE staining were performed, and the levels of osteogenic protein RUNX2 and osteogenesis-related factor VGLL4 were determined. Co-immunoprecipitation (Co-IP) was further employed to delve into the effects of BSD on the interactions between TEAD4 and RUNX2. The key osteogenic factors 1ALP, COLl1A1, and Osterix expression were detected by RT-qPCR. Co-IP and proximity ligation assay (PLA) were employed to scrutinize the influence of BSD on TEAD4 and RUNX2 inter-binding. Moreover, VGLL4 knockdown in BMSCs was conducted to confirm the role of VGLL4 in the therapeutic mechanism of BSD. RESULTS: BSD showed a dose-dependent protective effect against osteoporotic injury, as evidenced by improvement in bone volume, bone microarchitecture, and histomorphometry. Additionally, BSD treatment increased the levels of RUNX2 and its downstream target genes including ALP, COL1A1, and Osterix. Moreover, BSD upregulated VGLL4 expression and lessened TEAD4-RUNX2 interactions. In BMSCs experiment, BSD-containing serum could promote osteogenic differentiation of BMSCs, boosted the expression of osteogenesis-related factors and VGLL4 level. The knockdown of VGLL4 in BMSCs diminished the promotion effect of BSD in osteoblast differentiation, suggesting that VGLL4 play a vital role in the therapeutic effects exerted by BSD. CONCLUSION: BSD ameliorated osteoporosis injury and promoted osteoblast differentiation through upregulation of VGLL4 levels, which in turn antagonized TEAD4-mediated RUNX2 transcriptional repression. Our study implied that BSD may be an osteoporosis therapeutic agent.

Enoyl coenzyme a hydratase 1 attenuates aortic valve calcification by suppressing Runx2 via Wnt5a/Ca2+ pathway.

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.

Qianggu Decoction Alleviated Osteoporosis by Promoting Osteogenesis of BMSCs through Mettl3-Mediated m6A Methylation.

Osteoporosis development is linked to abnormal bone marrow mesenchymal stem cells (BMSCs) differentiation. N6-methyladenosine (m6A), a prevalent mRNA modification, is known to influence BMSCs' osteogenic capacity. Qianggu decoction (QGD), a traditional Chinese medicine for osteoporosis, has unknown effects on BMSCs differentiation. This study investigates QGD's impact on BMSCs and its potential to ameliorate osteoporosis through m6A regulation. Using Sprague-Dawley (SD) rats with ovariectomy-induced osteoporosis, it is evaluated QGD's antiosteoporotic effects through micro-CT, histology, Western blotting, and osteoblastogenesis markers. QGD is found to enhance bone tissue growth and upregulate osteogenic markers Runx2, OPN, and OCN. It also promoted BMSCs osteogenic differentiation, as shown by increased calcium nodules and ALP activity. QGD treatment significantly increased m6A RNA levels and Mettl3 expression in BMSCs. Silencing Mettl3 with siRNA negated QGD's osteogenic effects. Collectively, QGD may improve BMSCs differentiation and mitigate osteoporosis, potentially through Mettl3-mediated m6A modification.

Disruption of the creb3l1 gene causes defects in caudal fin regeneration and patterning in zebrafish Danio rerio.

BACKGROUND: The gene cAMP-Responsive Element Binding protein 3-like-1 (CREB3L1) has been implicated in bone development in mice, with CREB3L1 knock-out mice exhibiting fragile bones, and in humans, with CREB3L1 mutations linked to osteogenesis imperfecta. However, the mechanism through which Creb3l1 regulates bone development is not fully understood. RESULTS: To probe the role of Creb3l1 in organismal physiology, we used CRISPR-Cas9 genome editing to generate a Danio rerio (zebrafish) model of Creb3l1 deficiency. In contrast to mammalian phenotypes, the Creb3l1 deficient fish do not display abnormalities in osteogenesis, except for a decrease in the bifurcation pattern of caudal fin. Both, skeletal morphology and overall bone density appear normal in the mutant fish. However, the regeneration of caudal fin postamputation is significantly affected, with decreased overall regenerate and mineralized bone area. Moreover, the mutant fish exhibit a severe patterning defect during regeneration, with a significant decrease in bifurcation complexity of the fin rays and distalization of the bifurcation sites. Analysis of genes implicated in bone development showed aberrant patterning of shha and ptch2 in Creb3l1 deficient fish, linking Creb3l1 with Sonic Hedgehog signaling during fin regeneration. CONCLUSIONS: Our results uncover a novel role for Creb3l1 in regulating tissue growth and patterning during regeneration.

Hakai, a novel Runx2 interacting protein, augments osteoblast differentiation by rescuing Runx2 from Smurf2-mediated proteasome degradation.

Runt-related transcription factor 2 (Runx2) is a key regulator of osteoblast differentiation and bone formation. In Runx2-deficient embryos, skeletal development ceases at the cartilage anlage stage. These embryos die of respiratory failure upon birth and display a complete absence of bone and cartilage mineralization. Here, we identified Hakai, a type of E3 ubiquitin ligase as a potential Runx2 interacting partner through affinity pulldown-based proteomic approach. Subsequently, we observed that similar to Runx2, Hakai was downregulated in osteopenic ovariectomized rats, suggesting its involvement in bone formation. Consistent with this observation, Hakai overexpression significantly enhanced osteoblast differentiation in mesenchyme-like C3H10T1/2 as well as primary rat calvaria osteoblast (RCO) cells in vitro. Conversely, overexpression of a catalytically inactive Hakai mutant (C109A) exhibited minimal to no effect, whereas Hakai depletion markedly reduced endogenous Runx2 levels and impaired osteogenic differentiation in both C3H10T1/2 and RCOs. Mechanistically, Hakai physically interacts with Runx2 and enhances its protein turnover by rescuing it from Smad ubiquitination regulatory factor 2 (Smurf2)-mediated proteasome degradation. Wild-type Hakai but not Hakai-C109A inhibited Smurf2 protein levels through proteasome-mediated degradation. These findings underscore Hakai's functional role in bone formation, primarily through its positive modulation of Runx2 protein turnover by protecting it from Smurf2-mediated ubiquitin-proteasomal degradation. Collectively, our results demonstrate Hakai as a promising novel therapeutic target for osteoporosis.

Target analysis and identification of curcumin against vascular calcification.

To investigate the mechanism of curcumin (CUR) on vascular calcification (VC), we screen for common targets of CUR and atherosclerosis and verify the targets genes in vivo and in vitro experiments. The common targets of CUR and AS were screened and obtained using different databases. These target genes were analyzed by GO and KEGG pathway enrichment analysis. PPI network analysis was performed and to analyze the key targets. A rat VC model was constructed and CUR was fed for three weeks. The changes of vascular structure and calcium salt deposition were observed in H&E and Von Kossa staining. Further, the expression of these target proteins was detected in the primary VSMCs of VC. The 31 common targets were obtained. GO functional enrichment analysis obtained 1284 terms and KEGG pathway enriched 66 pathways. The key genes were identified in the cytoHubba plugin. The molecular docking analysis showed that CUR bound strongly to EGFR, STAT3 and BCL2. The animal experiments showed the deposition calcium salt reduced by the CUR administration. These proteins BMP2, RUNX2, EGFR, STAT3 and BAX expression were upregulated in VC group and CUR attenuated the upregulated expression. The signal protein Akt and p65 expression increased in VC group and decreased in CUR group. We identified some common target genes of CUR and AS and identified these key genes. The anti-VC effect of CUR was associated with the inhibition of upregulation of EGFR, STAT3 and RUNX2 expression in VSMCs.

Hypomethylation of the RUNX2 Gene Is a New Potential Biomarker of Primary Osteoporosis in Men and Women.

The search for the molecular markers of osteoporosis (OP), based on the analysis of differential deoxyribonucleic acid (DNA) methylation in bone cells and peripheral blood cells, is promising for developments in the field of the early diagnosis and targeted therapy of the disease. The Runt-related transcription factor 2 (RUNX2) gene is one of the key genes of bone metabolism, which is of interest in the search for epigenetic signatures and aberrations associated with the risk of developing OP. Based on pyrosequencing, the analysis of the RUNX2 methylation profile from a pool of peripheral blood cells in men and women over 50 years of age of Russian ethnicity from the Volga-Ural region of Russia was carried out. The level of DNA methylation in three CpG sites of the RUNX2 gene was assessed and statistically significant hypomethylation was revealed in all three studied CpG sites in men (U = 746.5, p = 0.004; U = 784, p = 0.01; U = 788.5, p = 0.01, respectively) and in one CpG site in women (U = 537, p = 0.03) with primary OP compared with control. In the general sample, associations were preserved for the first CpG site (U = 2561, p = 0.0001766). The results were obtained for the first time and indicate the existence of potentially new epigenetic signatures of RUNX2 in individuals with OP.

TFEB/3 Govern Repair Schwann Cell Generation and Function Following Peripheral Nerve Injury.

TFEB and TFE3 (TFEB/3), key regulators of lysosomal biogenesis and autophagy, play diverse roles depending on cell type. This study highlights a hitherto unrecognized role of TFEB/3 crucial for peripheral nerve repair. Specifically, they promote the generation of progenitor-like repair Schwann cells after axonal injury. In Schwann cell-specific TFEB/3 double knock-out mice of either sex, the TFEB/3 loss disrupts the transcriptomic reprogramming that is essential for the formation of repair Schwann cells. Consequently, mutant mice fail to populate the injured nerve with repair Schwann cells and exhibit defects in axon regrowth, target reinnervation, and functional recovery. TFEB/3 deficiency inhibits the expression of injury-responsive repair Schwann cell genes, despite the continued expression of c-jun, a previously identified regulator of repair Schwann cell function. TFEB/3 binding motifs are enriched in the enhancer regions of injury-responsive genes, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired despite TFEB/3 deficiency. These findings underscore a unique role of TFEB/3 in adult Schwann cells that is required for proper peripheral nerve regeneration.

Biocompatibility of Subperiosteal Dental Implants: Changes in the Expression of Osteogenesis-Related Genes in Osteoblasts Exposed to Differently Treated Titanium Surfaces.

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, ß-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.

ALKBH5-mediated m6A demethylation of Runx2 mRNA promotes extracellular matrix degradation and intervertebral disc degeneration.

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. CONCLUSION: 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.

Hdac3-deficiency increases senescence-associated distention of satellite DNA and telomere-associated foci in osteoprogenitor cells.

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 nonproliferative 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 with controls under osteogenic conditions. We then measured senescence-associated distention of satellite (SADS) DNA 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- WT BMSCs. Senolytics reduced viable cell numbers in Hdac3 CKOOsx BMSC cultures. These data demonstrate that the 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 such as 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 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.

Notoginsenoside R1 (NGR1) regulates the AGE-RAGE signaling pathway by inhibiting RUNX2 expression to accelerate ferroptosis in breast cancer cells.

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.