Exploring epigenetic strategies for the treatment of osteoporosis.

The worldwide trend toward an aging population has resulted in a higher incidence of chronic conditions, such as osteoporosis. Osteoporosis, a prevalent skeletal disorder characterized by decreased bone mass and increased fracture risk, encompasses primary and secondary forms, each with distinct etiologies. Mechanistically, osteoporosis involves an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Current pharmacological interventions for osteoporosis, such as bisphosphonates, denosumab, and teriparatide, aim to modulate bone turnover and preserve bone density. Hormone replacement therapy and lifestyle modifications are also recommended to manage the condition. While current medications offer therapeutic options, they are not devoid of limitations. Recent studies have highlighted the importance of epigenetic mechanisms, including DNA methylation and histone modifications, in regulating gene expression during bone remodeling. The use of epigenetic drugs, or epidrugs, to target these mechanisms offers a promising avenue for therapeutic intervention in osteoporosis. In this review, we comprehensively examine the recent advancements in the application of epidrugs for treating osteoporosis.

Dimethyl alpha-ketoglutarate inhibits proliferation in diffuse intrinsic pontine glioma by reprogramming epigenetic and transcriptional networks.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with limited therapeutic options. Here, we investigated the potential of dimethyl alpha-ketoglutarate (DMKG) as an anti-proliferative agent against DIPG and unraveled its underlying molecular mechanisms. DMKG exhibited robust inhibition of DIPG cell proliferation, colony formation, and neurosphere growth. Transcriptomic analysis revealed substantial alterations in gene expression, with upregulated genes enriched in hypoxia-related pathways and downregulated genes associated with cell division and the mitotic cell cycle. Notably, DMKG induced G1/S phase cell cycle arrest and downregulated histone H3 lysine 27 acetylation (H3K27ac) without affecting H3 methylation levels. The inhibition of AKT and ERK signaling pathways by DMKG coincided with decreased expression of the CBP/p300 coactivator. Importantly, we identified the c-MYC-p300/ATF1-p300 axis as a key mediator of DMKG's effects, demonstrating reduced binding to target gene promoters and decreased H3K27ac levels. Depletion of c-MYC or ATF1 effectively inhibited DIPG cell growth. These findings highlight the potent anti-proliferative properties of DMKG, its impact on epigenetic modifications, and the involvement of the c-MYC-p300/ATF1-p300 axis in DIPG, shedding light on potential therapeutic strategies for this devastating disease.

TMBIM6 deficiency leads to bone loss by accelerating osteoclastogenesis.

TMBIM6 is an endoplasmic reticulum (ER) protein that modulates various physiological and pathological processes, including metabolism and cancer. However, its involvement in bone remodeling has not been investigated. In this study, we demonstrate that TMBIM6 serves as a crucial negative regulator of osteoclast differentiation, a process essential for bone remodeling. Our investigation of Tmbim6-knockout mice revealed an osteoporotic phenotype, and knockdown of Tmbim6 inhibited the formation of multinucleated tartrate-resistant acid phosphatase-positive cells, which are characteristic of osteoclasts. Transcriptome and immunoblot analyses uncovered that TMBIM6 exerts its inhibitory effect on osteoclastogenesis by scavenging reactive oxygen species and preventing p65 nuclear localization. Additionally, TMBIM6 depletion was found to promote p65 localization to osteoclast-related gene promoters. Notably, treatment with N-acetyl cysteine, an antioxidant, impeded the osteoclastogenesis induced by TMBIM6-depleted cells, supporting the role of TMBIM6 in redox regulation. Furthermore, we discovered that TMBIM6 controls redox regulation via NRF2 signaling pathways. Our findings establish TMBIM6 as a critical regulator of osteoclastogenesis and suggest its potential as a therapeutic target for the treatment of osteoporosis.

The role of histone modifications: from neurodevelopment to neurodiseases.

Epigenetic regulatory mechanisms, including DNA methylation, histone modification, chromatin remodeling, and microRNA expression, play critical roles in cell differentiation and organ development through spatial and temporal gene regulation. Neurogenesis is a sophisticated and complex process by which neural stem cells differentiate into specialized brain cell types at specific times and regions of the brain. A growing body of evidence suggests that epigenetic mechanisms, such as histone modifications, allow the fine-tuning and coordination of spatiotemporal gene expressions during neurogenesis. Aberrant histone modifications contribute to the development of neurodegenerative and neuropsychiatric diseases. Herein, recent progress in understanding histone modifications in regulating embryonic and adult neurogenesis is comprehensively reviewed. The histone modifications implicated in neurodegenerative and neuropsychiatric diseases are also covered, and future directions in this area are provided.

Gene regulation by histone-modifying enzymes under hypoxic conditions: a focus on histone methylation and acetylation.

Oxygen, which is necessary for sustaining energy metabolism, is consumed in many biochemical reactions in eukaryotes. When the oxygen supply is insufficient for maintaining multiple homeostatic states at the cellular level, cells are subjected to hypoxic stress. Hypoxia induces adaptive cellular responses mainly through hypoxia-inducible factors (HIFs), which are stabilized and modulate the transcription of various hypoxia-related genes. In addition, many epigenetic regulators, such as DNA methylation, histone modification, histone variants, and adenosine triphosphate-dependent chromatin remodeling factors, play key roles in gene expression. In particular, hypoxic stress influences the activity and gene expression of histone-modifying enzymes, which controls the posttranslational modification of HIFs and histones. This review covers how histone methylation and histone acetylation enzymes modify histone and nonhistone proteins under hypoxic conditions and surveys the impact of epigenetic modifications on gene expression. In addition, future directions in this area are discussed.

The KDM4B-CCAR1-MED1 axis is a critical regulator of osteoclast differentiation and bone homeostasis.

Bone undergoes a constant and continuous remodeling process that is tightly regulated by the coordinated and sequential actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Recent studies have shown that histone demethylases are implicated in osteoblastogenesis; however, little is known about the role of histone demethylases in osteoclast formation. Here, we identified KDM4B as an epigenetic regulator of osteoclast differentiation. Knockdown of KDM4B significantly blocked the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. Mice with myeloid-specific conditional knockout of KDM4B showed an osteopetrotic phenotype due to osteoclast deficiency. Biochemical analysis revealed that KDM4B physically and functionally associates with CCAR1 and MED1 in a complex. Using genome-wide chromatin immunoprecipitation (ChIP)-sequencing, we revealed that the KDM4B-CCAR1-MED1 complex is localized to the promoters of several osteoclast-related genes upon receptor activator of NF-κB ligand stimulation. We demonstrated that the KDM4B-CCAR1-MED1 signaling axis induces changes in chromatin structure (euchromatinization) near the promoters of osteoclast-related genes through H3K9 demethylation, leading to NF-κB p65 recruitment via a direct interaction between KDM4B and p65. Finally, small molecule inhibition of KDM4B activity impeded bone loss in an ovariectomized mouse model. Taken together, our findings establish KDM4B as a critical regulator of osteoclastogenesis, providing a potential therapeutic target for osteoporosis.

Transcriptome profiling of olive flounder responses under acute and chronic heat stress.

BACKGROUND: The olive flounder (Paralichthys olivaceus) is a saltwater fish, which is valuable to the economy. The olive flounder strives to adapt to environmental stressors through physiological, biochemical, and transcriptional responses. The rise in water temperature threatens the growth, development, reproduction, and survival of olive flounder. Each organ in the olive flounder can differentially respond to heat stress. OBJECTIVES: The purpose of this study is to investigate organ-specific transcriptional changes in olive flounder tissues during heat stress. METHODS: In this study, transcriptome dynamics of the gill, liver, and muscle of olive flounder to acute or chronic heat stress were investigated. RESULTS: Principal component analysis plotting revealed that the transcriptome of each organ is quite separated. K-means clustering, gene ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analysis showed the differential transcriptome responses of each organ to heat stress. Heat stress commonly affects the pathways involved in the correct protein folding, DNA repair, and cell cycle. CONCLUSION: Our results may provide a valuable molecular basis of heat acclimation in fishes.

New Insights into the Role of Histone Changes in Aging.

Aging is the progressive decline or loss of function at the cellular, tissue, and organismal levels that ultimately leads to death. A number of external and internal factors, including diet, exercise, metabolic dysfunction, genome instability, and epigenetic imbalance, affect the lifespan of an organism. These aging factors regulate transcriptome changes related to the aging process through chromatin remodeling. Many epigenetic regulators, such as histone modification, histone variants, and ATP-dependent chromatin remodeling factors, play roles in chromatin reorganization. The key to understanding the role of gene regulatory networks in aging lies in characterizing the epigenetic regulators responsible for reorganizing and potentiating particular chromatin structures. This review covers epigenetic studies on aging, discusses the impact of epigenetic modifications on gene expression, and provides future directions in this area.

Transcriptome Analysis Reveals That Abeliophyllum distichum Nakai Extract Inhibits RANKL-Mediated Osteoclastogenensis Mainly Through Suppressing Nfatc1 Expression.

Abeliophyllum distichum Nakai is known as a monotypic genus endemic to South Korea. Currently, several pharmacological studies have revealed that A. distichum extract exhibits diverse biological functions, including anti-cancer, anti-diabetic, anti-hypertensive, and anti-inflammatory activities. In this study, we present the anti-osteoporotic activity of A. distichum extract by inhibiting osteoclast formation. First, we show that the methanolic extract of the leaves of A. distichum, but not extracts of the branches or fruits, significantly inhibits receptor activator of the NF-κB ligand (RANKL)-induced osteoclast differentiation. Second, our transcriptome analysis revealed that the leaf extract (LE) blocks sets of RANKL-mediated osteoclast-related genes. Third, the LE attenuates the phosphorylation of extracellular signal-related kinase. Finally, treatment with the LE effectively prevents postmenopausal bone loss in ovariectomized mice and glucocorticoid-induced osteoporosis in zebrafish. Our findings show that the extract of A. distichum efficiently suppressed osteoclastogenesis by regulating osteoclast-related genes, thus offering a novel therapeutic strategy for osteoporosis.

Ethyl Acetate Fraction of Aqueous Extract of Lentinula edodes Inhibits Osteoclastogenesis by Suppressing NFATc1 Expression.

Bone tissue is continuously remodeled by the coordinated action of osteoclasts and osteoblasts. Nuclear factor-activated T cells c1 (NFATc1) is a well-known transcription factor for osteoclastogenesis and transcriptionally activated by the c-Fos and nuclear factor-kappa B (NF-κB) signaling pathways in response to receptor activation of NF-κB ligand (RANKL). Since excessive RANKL signaling causes an increase of osteoclast formation and bone resorption, inhibition of RANKL or its signaling pathway is an attractive therapeutic approach to the treatment of pathologic bone loss. In this study, we show that an ethyl acetate fraction (LEA) from the shiitake mushroom, Lentinula edodes, inhibited RANKL-induced osteoclast differentiation by blocking the NFATc1 signaling pathway. We found that the water extract and its subsequent ethyl acetate fraction of L. edodes significantly suppressed osteoclast formation. Comparative transcriptome analysis revealed that LEA specifically downregulated a set of RANKL target genes, including Nfatc1. Next, we found that LEA suppresses Nfatc1 expression mainly through the inhibition of the transactivity of p65 and NFATc1. Moreover, treatment of LEA rescued an osteoporotic phenotype in a zebrafish model of glucocorticoid-induced osteoporosis. Collectively, our findings define an undocumented role of the shiitake mushroom extract in regulating bone development.

Tetracycline Analogs Inhibit Osteoclast Differentiation by Suppressing MMP-9-Mediated Histone H3 Cleavage.

Osteoporosis is a common disorder of bone remodeling, caused by the imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Recently, we reported that matrix metalloproteinase-9 (MMP-9)-dependent histone H3 proteolysis is a key event for proficient osteoclast formation. Although it has been reported that several MMP-9 inhibitors, such as tetracycline and its derivatives, show an inhibitory effect on osteoclastogenesis, the molecular mechanisms for this are not fully understood. Here we show that tetracycline analogs, especially tigecycline and minocycline, inhibit osteoclast formation by blocking MMP-9-mediated histone H3 tail cleavage. Our molecular docking approach found that tigecycline and minocycline are the most potent inhibitors of MMP-9. We also observed that both inhibitors significantly inhibited H3 tail cleavage by MMP-9 in vitro. These compounds inhibited receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast formation by blocking the NFATc1 signaling pathway. Furthermore, MMP-9-mediated H3 tail cleavage during osteoclast differentiation was selectively blocked by these compounds. Treatment with both tigecycline and minocycline rescued the osteoporotic phenotype induced by prednisolone in a zebrafish osteoporosis model. Our findings demonstrate that the tetracycline analogs suppress osteoclastogenesis via MMP-9-mediated H3 tail cleavage, and suggest that MMP-9 inhibition could offer a new strategy for the treatment of glucocorticoid-induced osteoporosis.

Bone Remodeling: Histone Modifications as Fate Determinants of Bone Cell Differentiation.

The bone tissue is a dynamic complex that constitutes of several interdependent systems and is continuously remodeled through the concerted actions of bone cells. Osteoblasts are mononucleated cells, derived from mesenchymal stem cells, responsible for bone formation. Osteoclasts are large multinucleated cells that differentiate from hematopoietic progenitors of the myeloid lineage and are responsible for bone resorption. The lineage-specific differentiation of bone cells requires an epigenetic regulation of gene expressions involving chromatin dynamics. The key step for understanding gene regulatory networks during bone cell development lies in characterizing the chromatin modifying enzymes responsible for reorganizing and potentiating particular chromatin structure. This review covers the histone-modifying enzymes involved in bone development, discusses the impact of enzymes on gene expression, and provides future directions and clinical significance in this area.

ß2-Adrenergic receptor (ß2-AR) agonist formoterol suppresses differentiation of L6 myogenic cells by blocking PI3K-AKT pathway.

ß2-Adrenergic receptor (ß2-AR) is implicated in muscle metabolic activities such as glycogen metabolism, glucose uptake, lipolysis and muscle growth. However, the functional role of ß2-AR in the differentiation of skeletal muscle is largely unknown. Here, we examined the functional role of ß2-AR in L6 myoblast differentiation using the long-term-acting ß2-AR-specific agonist formoterol. We observed that formoterol treatment strongly suppressed L6 myoblast differentiation and the expression of myosin heavy chain (MHC) in a dose- and time-dependent manner. Showing that both long-acting agonist (formoterol) and short-acting agonist (terbutaline) inhibited the induction of MHC protein, whereas ß2-AR antagonist (ICI-118,551) upregulated MHC expression, we clearly demonstrated that ß2-AR is involved in L6 myoblast differentiation. Furthermore, our pharmacological inhibition study revealed that the PI3K-AKT pathway is the main signaling pathway for myotube formation. Formoterol inhibited the activation of PI3K-AKT signaling, but not that of ERK signaling. Moreover, formoterol selectively inhibited AKT activation by IGF-I, but not by insulin. Collectively, our findings reveal a previously undocumented role of ß2-AR activation in modulating the differentiation of L6 myoblasts.

Carboxy-terminal domain of Cas differentially modulates c-Jun expression, DNA synthesis, and membrane ruffling induced by insulin, EGF, and IGF-1.

p130 Crk-associated substrate (Cas) is an adaptor protein associating with many other signaling proteins and regulates a various biological processes including cell adhesion, migration, and growth factor stimulation. However, the exact functional role of Cas in growth factor signaling pathway was poorly understood. Here we investigated the role of Cas and its domains in the effects of insulin, EGF, and IGF-1 on c-Jun gene expression, DNA synthesis, cytoskeletal reorganization. We found that microinjection of anti-Cas antibody and C-terminal domain of Cas (Cas-CT) specifically inhibited EGF-induced, but not insulin- or IGF-1-induced, c-Jun expression. Cell cycle progression and cytoskeleton reorganization induced by insulin and EGF, but not by IGF-1, were inhibited by microinjected anti-Cas and Cas-CT. In contrast, microinjection of the substate domain (Cas-SD) of Cas did not have any inhibitory effects. These results revealed that the Cas-CT is differentially implicated in insulin and EGF-mediated, but not IGF-1-mediated, c-Jun expression, DNA synthesis and membrane ruffling.

Oncogenic N-Ras Stimulates SRF-Mediated Transactivation via H3 Acetylation at Lysine 9.

Signal transduction pathways regulate the gene expression by altering chromatin dynamics in response to mitogens. Ras proteins are key regulators linking extracellular stimuli to a diverse range of biological responses associated with gene regulation. In mammals, the three ras genes encode four Ras protein isoforms: H-Ras, K-Ras4A, K-Ras4B, and N-Ras. Although emerging evidence suggests that Ras isoforms differentially regulate gene expressions and are functionally nonredundant, the mechanisms underlying Ras specificity and Ras signaling effects on gene expression remain unclear. Here, we show that oncogenic N-Ras acts as the most potent regulator of SRF-, NF-κB-, and AP-1-dependent transcription. N-Ras-RGL2 axis is a distinct signaling pathway for SRF target gene expression such as Egr1 and JunB, as RGL2 Ras binding domain (RBD) significantly impaired oncogenic N-Ras-induced SRE activation. By monitoring the effect of Ras isoforms upon the change of global histone modifications in oncogenic Ras-overexpressed cells, we discovered that oncogenic N-Ras elevates H3K9ac/H3K23ac levels globally in the chromatin context. Importantly, chromatin immunoprecipitation (ChIP) assays revealed that H3K9ac is significantly enriched at the promoter and coding regions of Egr1 and JunB. Collectively, our findings define an undocumented role of N-Ras in modulating of H3 acetylation and in gene regulation.

Histone tail cleavage as a novel epigenetic regulatory mechanism for gene expression.

Chromatin is an intelligent building block that can express either external or internal needs through structural changes. To date, three methods to change chromatin structure and regulate gene expression have been well-documented: histone modification, histone exchange, and ATP-dependent chromatin remodeling. Recently, a growing body of literature has suggested that histone tail cleavage is related to various cellular processes including stem cell differentiation, osteoclast differentiation, granulocyte differentiation, mammary gland differentiation, viral infection, aging, and yeast sporulation. Although the underlying mechanisms suggesting how histone cleavage affects gene expression in view of chromatin structure are only beginning to be understood, it is clear that this process is a novel transcriptional epigenetic mechanism involving chromatin dynamics. In this review, we describe the functional properties of the known histone tail cleavage with its proteolytic enzymes, discuss how histone cleavage impacts gene expression, and present future directions for this area of study. [BMB Reports 2018; 51(5): 211-218].

Functional roles of BCAR3 in the signaling pathways of insulin leading to DNA synthesis, membrane ruffling and GLUT4 translocation.

Breast cancer anti-estrogen resistance 3 (BCAR3) is an SH2-containing signal transducer and is implicated in tumorigenesis of breast cancer cells. In this study, we found that BCAR3 mediates the induction of ERK activation and DNA synthesis by insulin, but not by IGF-1. Specifically, the SH2 domain of BCAR3 is involved in insulin-stimulated DNA synthesis. Differential tyrosine-phosphorylated patterns of the BCAR3 immune complex were detected in insulin and IGF-1 signaling, suggesting that BCAR3 is a distinct target molecule of insulin and IGF-1 signaling. Moreover, microinjection of BCAR3 inhibitory materials inhibited membrane ruffling induced by insulin, while this did not affect insulin-mediated GLUT4 translocation. Taken together, these results demonstrated that BCAR3 plays an important role in the signaling pathways of insulin leading to cell cycle progression and cytoskeleton reorganization, but not GLUT4 translocation.

Expression of cassini, a murine gamma-satellite sequence conserved in evolution, is regulated in normal and malignant hematopoietic cells.

BACKGROUND: Acute lymphoblastic leukemia (ALL) cells treated with drugs can become drug-tolerant if co-cultured with protective stromal mouse embryonic fibroblasts (MEFs). RESULTS: We performed transcriptional profiling on these stromal fibroblasts to investigate if they were affected by the presence of drug-treated ALL cells. These mitotically inactivated MEFs showed few changes in gene expression, but a family of sequences of which transcription is significantly increased was identified. A sequence related to this family, which we named cassini, was selected for further characterization. We found that cassini was highly upregulated in drug-treated ALL cells. Analysis of RNAs from different normal mouse tissues showed that cassini expression is highest in spleen and thymus, and can be further enhanced in these organs by exposure of mice to bacterial endotoxin. Heat shock, but not other types of stress, significantly induced the transcription of this locus in ALL cells. Transient overexpression of cassini in human 293 embryonic kidney cells did not increase the cytotoxic or cytostatic effects of chemotherapeutic drugs but provided some protection. Database searches revealed that sequences highly homologous to cassini are present in rodents, apicomplexans, flatworms and primates, indicating that they are conserved in evolution. Moreover, CASSINI RNA was induced in human ALL cells treated with vincristine. Surprisingly, cassini belongs to the previously reported murine family of γ-satellite/major satellite DNA sequences, which were not known to be present in other species. CONCLUSIONS: Our results show that the transcription of at least one member of these sequences is regulated, suggesting that this has a function in normal and transformed immune cells. Expression of these sequences may protect cells when they are exposed to specific stress stimuli.