CDDO-Im ameliorates osteoarthritis and inhibits chondrocyte apoptosis in mice via enhancing Nrf2-dependent autophagy.

Osteoarthritis (OA) is the most prevalent chronic degenerative joint disease with few treatment options. The pathogenesis of OA is characterized by sustained inflammation, oxidative stress and chondrocyte apoptosis that eventually lead to cartilage degradation and joint dysfunction. In the present study, we identified a synthetic triterpenoid CDDO-Im(1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole) as an activator of Nrf2 (nuclear factor erythroid 2-related factor 2) that displayed strong anti-OA effects. We showed that CDDO-Im (20 nM) significantly alleviated TNF-α-induced apoptosis of primary human chondrocytes and extracellular matrix degradation. In a mouse OA model incurred by DMM (destabilization of medial meniscus), administration of CDDO-Im (2.5 mg/kg, ip, every other day for 8 weeks) effectively reduced knee joint cartilage erosion and serum levels of inflammatory cytokines IL-1ß and IL-6. We revealed that CDDO-Im (20 nM) significantly enhanced autophagy activities in chondrocytes, whereas the autophagy inhibition by chloroquine (CQ, 50 µM) or 3-methyladenine (3-MA, 5 mM) abrogated the anti-apoptosis and chondroprotective effects of CDDO-Im in TNF-α-treated chondrocytes. Moreover, we confirmed that CDDO-Im (1-20 nM) dose-dependently activated Nrf2 pathway in TNF-α-treated chondrocytes, and its chondroprotective and autophagy-enhancing effects were significantly diminished when Nrf2 signaling was blocked by Nrf2 inhibitor ML385 (20 µM) or siRNA-mediated Nrf2 knockdown. Together, our results demonstrate that CDDO-Im exhibits prominent chondroprotective and anti-OA activities owing to its Nrf2 activation and autophagy-enhancing properties, which might provide new insights into the strategies of OA clinical prevention and treatment.

Enhancing PPARγ by HDAC inhibition reduces foam cell formation and atherosclerosis in ApoE deficient mice.

Atherosclerosis (AS) is a risky cardiovascular disease with limited treatment options. Various pan or type-selective histone deacetylase (HDAC) inhibitors are reportedly atheroprotective against atherosclerosis (AS); however, the key effectors and the main cellular processes that mediate the protective effects remain poorly defined. Here, we report that PPARγ (Peroxisome proliferator-activated receptor gamma), a transcription factor actively involved in lipid metabolism with strong tissue protective and anti-inflammation properties, is a critical mediator of the anti-AS effects by HDAC inhibition. We showed that a well-known pan-HDAC inhibitor TSA (Trichostatin A) reduced foam cell formation of macrophages that is accompanied by a marked elevation of PPARγ and its downstream cholesterol efflux transporter ABCA1 (ATP-binding membrane cassette transport protein A1) and ABCG1. In an AS model of ApoE-/- mice fed on high-fat diet, TSA treatment alleviated AS lesions, similarly increased PPARγ and the downstream cholesterol transporters and mitigated the induction of inflammatory cytokine TNFα and IL-1ß. Exploring the potential cause of PPARγ elevation revealed that TSA induced the acetylation of C/EBPα (CCAAT enhancer binding protein alpha), the upstream regulator of PPARγ, through which it increased PPARγ transactivation. More importantly, we generated a strain of PPARγ/ApoE double knockout mice and demonstrated that lack of PPARγ abrogated the protective effects of TSA on foam cell formation of peritoneal macrophages and the AS pathogenesis. Taken together, these results unravel that C/EBPα and PPARγ are the HDAC-sensitive components of an epigenetic signaling pathway mediating foam cell formation and AS development, and suggest that targeting C/EBPα/PPARγ axis by HDAC inhibitors possesses therapeutic potentials in retarding the progression of AS and the related cardiovascular diseases.

Significant Down-Regulation of Urea Cycle Generates Clinically Relevant Proteomic Signature in Hepatocellular Carcinoma Patients with Macrovascular Invasion.

Vascular invasion is considered as the critical risk factor of hepatocellular carcinoma (HCC). To reveal the molecular mechanisms underlying macrovascular invasion (MaVI) in HCC, we performed an iTRAQ based proteomic study to identify notably dysregulated proteins from eight HCC patients with differential vascular invasion and further confirmed them in the other 53 HCC patients. Forty-seven proteins were found significantly down-regulated in HCC with MaVI. More importantly, 30 of them were not changed in HCC without MaVI. Gene ontology analysis of these 47 proteins shows the top three enriched biological processes are urea cycle, gluconeogenesis, and arginine biosynthetic process. We validated nine remarkably dysregulated candidates in HCC patients with MaVI by Western blot including eight down-regulated proteins (CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin A6, and CES1) and one up-regulated protein (CKAP4). Furthermore, dysregulation of CPS1, ASL, and ARG1, key enzymes involved in urea cycle, together with Annexin A6 and CES1, major proteins in regulating cholesterol homeostasis and fatty acid ester metabolism, was verified using immunohistochemical staining. The significant down-regulation of urea cycle generates clinically relevant proteomic signature in HCC patients with macrovascular invasion, which may provide possible insights into the molecular mechanisms of metastasis and new therapeutic targets of HCC.

PPARγ preservation via promoter demethylation alleviates osteoarthritis in mice.

OBJECTIVES: Osteoarthritis (OA) is the most common degenerative joint disease in aged population and its development is significantly influenced by aberrant epigenetic modifications of numerous OA susceptible genes; however, the precise mechanisms that DNA methylation alterations affect OA pathogenesis remain undefined. This study investigates the critical role of epigenetic PPARγ (peroxisome proliferator-activated receptor-gamma) suppression in OA development. METHODS: Articular cartilage expressions of PPARγ and bioactive DNA methyltransferases (DNMTs) from OA patients and mice incurred by DMM (destabilisation of medial meniscus) were examined. DNA methylation status of both human and mouse PPARγ promoters were assessed by methylated specific PCR and/or bisulfite-sequencing PCR. OA protections by a pharmacological DNA demethylating agent 5Aza (5-Aza-2'-deoxycytidine) were compared between wild type and PPARγ knockout mice. RESULTS: Articular cartilages from both OA patients and DMM mice display substantial PPARγ suppressions likely due to aberrant elevations of DNMT1 and DNMT3a and consequential PPARγ promoter hypermethylation. 5Aza known to inhibit both DNMT1 and DNMT3a reversed the PPARγ promoter hypermethylation, recovered the PPARγ loss and effectively attenuated the cartilage damage in OA mice. 5Aza also inhibited the OA-associated excessive inflammatory cytokines and deficit anti-oxidant enzymes, which were blocked by a specific PPARγ inhibitor in cultured chondrocytes. Further, 5Aza-confered protections against the cartilage damage and the associated abnormalities of OA-susceptible factors were significantly abrogated in PPARγ knockout mice. CONCLUSION: Epigenetic PPARγ suppression plays a key role in OA development and PPARγ preservation via promoter demethylation possesses promising therapeutic potentials in clinical treatment of OA and the related joint diseases.

TGFß-incurred epigenetic aberrations of miRNA and DNA methyltransferase suppress Klotho and potentiate renal fibrosis.

Renal fibrosis is a common pathological feature of chronic kidney diseases (CKD) and its development and progression are significantly affected by epigenetic modifications such as aberrant miRNA and DNA methylation. Klotho is an anti-aging and anti-fibrotic protein and its early decline after renal injury is reportedly associated with aberrant DNA methylation. However, the key upstream pathological mediators and the molecular cascade leading to epigenetic Klotho suppression are not exclusively established. Here we investigate the epigenetic mechanism of Klotho deficiency and its functional relevance in renal fibrogenesis. Fibrotic kidneys induced by unilateral ureteral occlusion (UUO) displayed marked Klotho suppression and the promoter hypermethylation. These abnormalities were likely due to deregulated transforming growth factor-beta (TGFß) since TGFß alone caused the similar epigenetic aberrations in cultured renal cells and TGFß blockade prevented the alterations in UUO kidney. Further investigation revealed that TGFß enhanced DNA methyltransferase (DNMT) 1 and DNMT3a via inhibiting miR-152 and miR-30a in both renal cells and fibrotic kidneys. Accordingly the blockade of either TGFß signaling or DNMT1/3a activities significantly recovered the Klotho loss and attenuated pro-fibrotic protein expression and renal fibrosis. Moreover, Klotho knockdown by RNA interferences abolished the anti-fibrotic effects of DNMT inhibition in both TGFß-treated renal cell and UUO kidney, indicating that TGFß-mediated miR-152/30a inhibitions, DNMT1/3a aberrations and subsequent Klotho loss constitute a critical regulatory loop that eliminates Klotho's anti-fibrotic activities and potentiates renal fibrogenesis. Thus, our study elaborates a novel epigenetic cascade of renal fibrogenesis and reveals the potential therapeutic targets for treating the renal fibrosis-associated kidney diseases.

Rhein reverses Klotho repression via promoter demethylation and protects against kidney and bone injuries in mice with chronic kidney disease.

Rhein is an anthraquinone compound isolated from the medicinal plant rhubarb and mainly used in the clinical treatment of diabetic nephropathy. Rhein exhibits various renoprotective functions, but the underlying mechanisms are not fully determined. However, its renoprotective properties recapitulate the role of Klotho, a renal-specific antiaging protein critical for maintaining kidney homeostasis. Here we explored the connections between rhein renoprotection and Klotho in a mouse model of adenine-induced chronic kidney disease. In addition to being an impressive Klotho upregulator, rhein remarkably reversed renal Klotho deficiency in adenine-treated mice. This effect was associated with significant improvement in disturbed serum biochemistry, profibrogenic protein expression, and kidney and bone damage. Further investigation of the molecular basis of Klotho loss revealed that these kidneys displayed marked inductions of DNA methyltransferase DNMT1/DNMT3a and Klotho promoter hypermethylation, whereas rhein treatment effectively corrected these alterations. The renal protective effects of rhein were largely abolished when Klotho was knocked-down by RNA interferences, suggesting that rhein reversal of Klotho deficiency is essential for its renoprotective actions. Thus, our study clarifies how rhein regulation of Klotho expression contributes to its renoprotection and brings new insights into Klotho-targeted strategy for the treatment of kidney diseases of various etiologies.

Klotho restoration via acetylation of Peroxisome Proliferation-Activated Receptor γ reduces the progression of chronic kidney disease.

Klotho is an anti-aging protein mainly expressed in the kidney. Reduced Klotho expression closely correlates with the development and progression of chronic kidney disease (CKD). Klotho is also a downstream gene of Peroxisome Proliferation-Activated Receptor γ (PPARγ), a major transcription factor whose functions are significantly affected by post-translational modifications including acetylation. However, whether PPARγ acetylation regulates renal Klotho expression and function in CKD is unknown. Here we test whether renal damage and reduced Klotho expression in the adenine CKD mouse model can be attenuated by the pan histone deacetylase (HDAC) inhibitor trichostatin A. This inhibition up-regulated Klotho mainly through an enhancement of PPARγ acetylation, stimulation of PPARγ binding to Klotho promoter, and PPARγ-dependent increase in Klotho transcription, with a substantial control of the regulation occurring via PPARγ acetylations on K240 and K265. Consistently trichostatin A-induced reversal of Klotho loss and renoprotective effects were abrogated in PPARγ knockout mice, supporting that PPARγ is an essential acetylation target for Klotho restoration and renal protection. Intriguingly, the kidneys of adenine-fed CKD mice displayed deregulated HDAC3 up-regulation. Selective HDAC3 inhibition effectively alleviated Klotho loss and kidney injury, whereas the protective effects were largely abolished when Klotho was knocked down by siRNA, suggesting that aberrant HDAC3 and Klotho loss are crucial components involved in the renal damage of mice with CKD. Our study identified an important signaling cascade and key components contributing to the pathogenesis of CKD. Thus, targeting Klotho loss by HDAC3 inhibition has promising therapeutic potential for the reduction of CKD progression.

Sinomenine attenuates renal fibrosis through Nrf2-mediated inhibition of oxidative stress and TGFß signaling.

Renal fibrosis is the common feature of chronic kidney disease and mainly mediated by TGFß-associated pro-fibrogenic signaling, which causes excessive extracellular matrix accumulation and successive loss of kidney functions. Sinomenine (SIN), an alkaloid derived from medicinal herb extensively used in treatment of rheumatoid arthritis and various inflammatory disorders, displays renal protective properties in experimental animals; however its pharmacological potency against renal fibrosis is not explored. In this study we report that SIN possesses strong anti-renal fibrosis functions in kidney cell and in mouse fibrotic kidney. SIN beneficially modulated the pro-fibrogenic protein expression in TGFß-treated kidney cells and attenuated the renal fibrotic pathogenesis incurred by unilateral ureteral obstruction (UUO), which correlated with its activation of Nrf2 signaling - the key defender against oxidative stress with anti-fibrotic potentials. Further investigation on its regulation of Nrf2 downstream events revealed that SIN significantly balanced oxidative stress via improving the expression and activity of anti-oxidant and detoxifying enzymes, and interrupted the pro-fibrogenic signaling of TGFß/Smad and Wnt/ß-catenin. Even more impressively SIN achieved its anti-fibrotic activities in an Nrf2-dependent manner, suggesting that SIN regulation of Nrf2-associated anti-fibrotic activities constitutes a critical component of SIN's renoprotective functions. Collectively our studies have demonstrated a novel anti-fibrotic property of SIN and its upstream events and provided a molecular basis for SIN's potential applications in treatment of renal fibrosis-associated kidney disorders.

Nrf2 is essential for the anti-inflammatory effect of carbon monoxide in LPS-induced inflammation.

INTRODUCTION: Carbon monoxide (CO) released from CORM-2 has anti-inflammatory function, but the critical molecule mediating the inflammation inhibition has not been elucidated. Previous studies indicate that CORM-2 can activate Nrf2, a key transcription factor regulating host defense against oxidative stress and inflammation-related disorders. In this study we use Nrf2 knockout mice to determine the role of Nrf2 in mediating the CO anti-inflammatory action. METHODS: We compared CORM-2's inhibiting effect on pro-inflammatory cytokine expressions (TNF-α, IL-1ß and IL-6 and iNOS) in primary peritoneal macrophages, mouse liver and brain tissues from Nrf2(+/+) and Nrf2(-/-) mice. We further assayed the inflammatory cell infiltration in both liver and brain tissues of the Nrf2(+/+) and Nrf2(-/-) mice. Finally, we examined CORM's influence on mouse mortality in a mouse sepsis model. RESULTS: Our results showed that CORM-2 dramatically inhibited the expression of pro-inflammatory cytokines in Nrf2(+/+) mice, but not in Nrf2(-/-) mice. Furthermore CORM-2 substantially decreased LPS-induced mouse mortality of Nrf2(+/+) mice, but not of Nrf2(-/-) mice. CONCLUSION: We conclude that Nrf2 is indispensable for CORM-2 inhibition of LPS-induced inflammation.

Longikaurin A induces ferroptosis and inhibits glioblastoma progression through DNA methylation - Mediated GPX4 suppression.

Glioblastoma (GBM) is the most common primary intracranial tumor highly resistant to conventional clinical chemotherapy. Recently, the induction of ferroptosis is emerging as a putative strategy to treat various tumors. However, the identification of the effective and applicable tumor ferroptosis-inducing agents remains challenging. In this study, we showed that longikaurin A (LK-A), a natural diterpenoid isolated from the medicinal plant Isodon ternifolius with strong anti-GBM capacities, induced remarkable GBM cell ferroptosis along with suppressing the key anti-ferroptosis factor glutathione peroxidase 4 (GPX4). GPX4 promoter contains conserved CpG islands. The LK-A-induced GPX4 suppression coincided with the inhibition of ten-eleven translocation 2 (TET2), a key DNA demethylation enzyme and an increase in the hypermethylation of the GPX4 promoter. Further, LK-A promoted the GBM ferroptotic alterations and inhibited GBM progression in both subcutaneous and orthotopic xenograft mouse models, whereas GPX4 overexpression largely abrogated its anti-GBM effects both in vitro and in vivo, suggesting that LK-A inductions of the DNA methylation-incurred GPX4 suppression and ferroptosis are crucial for its anti-GBM functions. Together, our study has elaborated an important epigenetic pathway of GBM ferroptosis and uncovered a critical pharmacological property of LK-A for treating GBM patients.

DNA Methylation-Mediated GPX4 Transcriptional Repression and Osteoblast Ferroptosis Promote Titanium Particle-Induced Osteolysis.

Metal wear particles generated by the movement of joint prostheses inevitably lead to aseptic osteolytic damage and ultimately prosthesis loosening, which are aggravated by various types of regulated cell death of bone. Nevertheless, the exact cellular nature and regulatory network underlying osteoferroptosis are poorly understood. Here, we report that titanium particles (TP) induced severe peri-implant osteolysis and ferroptotic changes with concomitant transcriptional repression of a key anti-ferroptosis factor, GPX4, in a mouse model of calvarial osteolysis. GPX4 repression was accompanied by an increase in DNA methyltransferases (DNMTs) 1/3a/3b and hypermethylation of the Gpx4 promoter, which were partly mediated by the transcriptional regulator/co-repressor KLF5 and NCoR. Conversely, treatment with SGI-1027, a DNMT-specific inhibitor, resulted in marked reversal of Gpx4 promoter hypermethylation and GPX4 repression, as well as improvement in ferroptotic osteolysis to a similar extent as with a ferroptosis inhibitor, liproxstatin-1. This suggests that epigenetic GPX4 repression and ferroptosis caused by the increase of DNMT1/3a/3b have a causal influence on TP-induced osteolysis. In cultured primary osteoblasts and osteoclasts, GPX4 repression and ferroptotic changes were observed primarily in osteoblasts that were alleviated by SGI-1027 in a GPX4 inactivation-sensitive manner. Furthermore, we developed a mouse strain with Gpx4 haplodeficiency in osteoblasts (Gpx4 Ob+/-) that exhibited worsened ferroptotic osteolysis in control and TP-treated calvaria and largely abolished the anti-ferroptosis and osteoprotective effects of SGI-1027. Taken together, our results demonstrate that DNMT1/3a/3b elevation, resulting GPX4 repression, and osteoblastic ferroptosis form a critical epigenetic pathway that significantly contributes to TP-induced osteolysis, and that targeting DNMT aberration and the associated osteoferroptosis could be a potential strategy to prevent or slow down prosthesis-related osteolytic complications.

Loss of Claudin-1 incurred by DNMT aberration promotes pancreatic cancer progression.

Pancreatic cancer (PC) is one of the most malignant and deadly tumors of digestive system with complex etiology and pathogenesis. Dysregulations of oncogenes and tumor suppressors due to epigenetic modifications causally affect tumorogenesis; however the key tumor suppressors and their regulations in PC are only partially defined. In this study, we found that Claudin-1 (encoded by CLDN1 gene) was significantly suppressed in PC that correlated with a poor clinical prognosis. Claudin-1 knockdown enhanced PC cell proliferation, migration, and stemness. Pancreatic specific Cldn1 knockout in KPC (LSLKrasG12D/Pdx1-Cre/Trp53R172H+) and KC (LSLKrasG12D/Pdx1-Cre) mice reduced mouse survival, promoted acinar-to-ductal metaplasia (ADM) process, and accelerated the development of pancreatic intraepithelial neoplasia (PanIN) and PC. Further investigation revealed that Claudin-1 suppression was mainly caused by aberrant DNA methylatransferase 1 (DNMT1) and DNMT3A elevations and the resultant CLDN1 promoter hypermethylation, as a DNMT specific inhibitor SGI-1027 effectively reversed the Claudin-1 suppression and inhibited PC progression both in vitro and in vivo in a Claudin-1 preservation-dependent manner. Together, our data suggest that Claudin-1 functions as a tumor suppressor in PC and its epigenetic suppression due to DNMT aberrations is a crucial event that promotes PC development and progression.

Bromodomain-containing protein 4 activates androgen receptor transcription and promotes ovarian fibrosis in PCOS.

Polycystic ovary syndrome (PCOS) is an endocrine disorder and the main cause of anovulatory infertility, in which persistent activation of androgen receptor (AR) due to aberrant acetylation modifications of transcription is a potential trigger; however, the precise mechanisms of AR activation are poorly understood. In this study, AR activation in dehydroepiandrosterone- and letrozole-induced rat PCOS ovaries coincided with a marked increase of a chromatin acetylation "reader" BRD4. Further bioinformatic analysis showed that the AR promoter contained highly conserved binding motifs of BRD4 and HIF-1α. BRD4 and HIF-1α inducibly bound to the histone 3/4 acetylation-modified AR promoter, while administration of a BRD4-selective inhibitor JQ1 reduced the binding and AR transcription and improved the adverse expression of the core fibrotic mediators in PCOS ovaries and DHT-treated granulosa cells. Our data indicate that BRD4 upregulation and the resultant AR transcriptional activation constitute an important regulatory pathway that promotes ovarian fibrosis in PCOS.

ZBTB7C m6A modification incurred by METTL3 aberration promotes osteosarcoma progression.

Aberrant N6-methyladenosine (m6A) modification of mRNAs contributes significantly to the epigenetic tumorigenesis, however, its precise role and the key targets in osteosarcoma (OS) are not defined. Here we reported that selective METTL3 (methyltransferase like 3) elevation and the consequential increase of m6A modification causally affect OS progression. The fast-growing OS cells displayed preferential upregulation of METTL3 and increased m6A modification. Conversely, m6A inhibition by 3-deazaadenosine, siRNA-mediated METTL3 knockdown or a METTL3-selective inhibitor STM2457 effectively inhibits OS cell growth and induced OS cell apoptosis. Further investigation revealed that an oncogenic protein ZBTB7C was likely a critical m6A target that mediated the oncogenic effects. ZBTB7C mRNA contains a typical m6A motif of high confidence and its mRNA and protein were enriched with increased m6A modification in OS samples/cells. In an OS xenograft model, STM2457 or siRNA-mediated METTL3 knockdown effectively lowed ZBTB7C abundance. More importantly, the anti-OS effects of STM2457 were significantly reduced when ZBTB7C was overexpressed by lentivirus. Together, our results demonstrate that the METTL3 aberration and the resultant ZBTB7C m6A modification form an important epigenetic regulatory loop that promotes OS progression, and targeting the METTL3/ZBTB7C axis may provide novel insights into the potential strategies for OS therapy.

HDAC3 aberration-incurred GPX4 suppression drives renal ferroptosis and AKI-CKD progression.

Acute kidney injury (AKI) progression to chronic kidney disease (CKD) represents a unique renal disease setting characterized by early renal cellular injury and regulated cell death, and later renal fibrosis, of which the critical role and nature of ferroptosis are only partially understood. Here, we report that renal tubular epithelial ferroptosis caused by HDAC3 (histone deacetylase 3) aberration and the resultant GPX4 suppression drives AKI-CKD progression. In mouse models of AKI-CKD transition induced by nephrotoxic aristolochic acid (AA) and folic acid (FA), renal tubular epithelial ferroptosis occurred early that coincided with preferential HDAC3 elevation and marked suppression of a core anti-ferroptosis enzyme GPX4 (glutathione peroxidase 4). Intriguingly, genetic Hdac3 knockout or administration of a HDAC3-selective inhibitor RGFP966 effectively mitigated the GPX4 suppression, ferroptosis and the fibrosis-associated renal functional loss. In cultured tubular epithelial cells, HDAC3 over-expression or inhibition inversely affected GPX4 abundances. Further analysis revealed that Gpx4 promoter contains a typical binding motif of transcription factor KLF5 (Kruppel-like factor 5). HDAC3 and KLF5 inducibly associated and bound to Gpx4 promoter upon AA treatment, leading to local histone hypoacetylation and GPX4 transactivation inhibition, which was blocked by RGFP966 and a KLF5 inhibitor ML264, respectively, suggesting that KLF5 co-regulated the HDAC3-incurred Gpx4 transcription inhibition. More importantly, in AKI-CKD mice receiving a GPX4 inactivator RSL3, the anti-ferroptosis and renoprotective effects of RGFP966 were largely abrogated, indicating that GPX4 is an essential downstream mediator of the HDAC3 aberration and renal ferroptosis during AKI-CKD transition. Together, our study identified a critical epigenetic pathway of ferroptosis during AKI-CKD transition and suggested that the strategies preserving GPX4 by HDAC3 inhibition are potentially effective to reduce renal ferroptosis and slow AKI-CKD progression.

Long noncoding RNA TUG1 promotes malignant progression of osteosarcoma by enhancing ZBTB7C expression.

BACKGROUND: Dysregulation of long non-coding RNAs (lncRNAs) is an important component of tumorigenesis. Aberrant expression of lncRNA taurine upregulated gene 1 (lncTUG1) has been reported in various tumors; however, its precise role and key targets critically involved in osteosarcoma (OS) progression remains unclear. METHODS: The expression profiles of lncRNAs and its regulated miRNAs related to OS progression were assessed by bioinformatics analysis and confirmed by qRT-PCR of OS cells. The miRNA targets were identified by transcriptome sequencing and verified by luciferase reporter and RNA pull-down assays. Several in vivo and in vitro approaches, including CCK8 assay, western blot, qRT-PCR, lentiviral transduction and OS cell xenograft mouse model were established to validate the effects of lncTUG1 regulation of miRNA and the downstream target genes on OS cell growth, apoptosis and progression. RESULTS: We found that lncTUG1 and miR-26a-5p were inversely up or down-regulated in OS cells, and siRNA-mediated lncTUG1 knockdown reversed the miR-26a-5p down-regulation and suppressed proliferation and enhanced apoptosis of OS cells. Further, we identified that an oncoprotein ZBTB7C was also upregulated in OS cells that were subjected to lncTUG1/miR-26a-5p regulation. More importantly, ZBTB7C knockdown reduced the ZBTB7C upregulation and ZBTB7C overexpression diminished the anti-OS effects of lncTUG1 knockdown in the OS xenograft model. CONCLUSIONS: Our data suggest that lncTUG1 acts as a miR-26a-5p sponge and promotes OS progression via up-regulating ZBTB7C, and targeting lncTUG1 might be an effective strategy to treat OS.

Targeted mutation of Fas ligand gene attenuates brain inflammation in experimental stroke.

Inflammation is an important contributing mechanism in ischemic brain injury. The current study elucidates a previously unexplored role of Fas ligand (FasL) in post-stroke inflammatory responses that is independent of its well-known effect in triggering apoptosis. Focal cerebral ischemia was induced for 2 h by right middle cerebral artery occlusion (MCAO) in FasL mutant (gld) and wild-type mice. FasL mutation profoundly reduced brain damage and improved neurological performance from 6 to 72 h after ischemic stroke. The production of inflammatory cytokines in the brain was attenuated in gld mice after ischemia in the absence of dramatic change in inflammatory cell apoptosis. FasL mutation attenuated the recruitment of peripheral inflammatory cells (neutrophil) and inhibited the activation of residential glial cells (microglia and astrocyte). FasL mutation reduced CD8(+) T cells and turned the Th1/Th2 balance towards Th2 in the brain and peripheral blood after cerebral ischemia. In contrast to cerebral ischemia, the molecular and cellular inflammatory changes induced by intracerebroventricular injection of lipopolysaccharide (LPS) were also attenuated in gld mice. Moreover, the soluble FasL (sFasL) and phospho-SAPK/JNK were decreased in gld mice, suggesting that the inflammatory role of FasL in experimental stroke might relate to sFasL and the c-Jun N-terminal kinase (JNK) signaling pathway. Taken together, our data suggest a novel role of FasL in the damaging inflammatory responses associated with cerebral ischemia. Neutralization of FasL may be a novel therapeutic strategy to suppress post-stroke inflammation and improve the long-term outcomes of stroke.

Histone deacetylase 3 (HDAC3) as an important epigenetic regulator of kidney diseases.

Development and progression of many kidney diseases are substantially influenced by aberrant protein acetylation modifications of gene expression crucial for kidney functions. Histone deacetylase (HDAC) expression alterations are detected from renal samples of patients and animal models of various kidney diseases, and the administrations of HDAC inhibitors display impressive renal protective effects in vitro and in vivo. However, when the expression alterations of multiple HDACs occur, not all the HDACs causally affect the disease onset or progression. Identification of a single HDAC as a disease-causing factor will allow subtype-targeted intervention with less side effect. HDAC3 is a unique HDAC with distinct structural and subcellular distribution features and co-repressor dependency. HDAC3 is required for kidney development and its aberrations actively participate in many pathological processes, such as cancer, cardiovascular diseases, diabetes, and neurodegenerative disorders, and contribute significantly to the pathogenesis of kidney diseases. This review will discuss the recent studies that investigate the critical roles of HDAC3 aberrations in kidney development, renal aging, renal cell carcinoma, renal fibrosis, chronic kidney disease, polycystic kidney disease, glomerular podocyte injury, and diabetic nephropathy. These studies reveal the distinct characters of HDAC3 aberrations that act on different molecules/signaling pathways under various renal pathological conditions, which might shed lights into the epigenetic mechanisms of renal diseases and the potentially therapeutic strategies.

Inhibition of DNA methyltransferase aberrations reinstates antioxidant aging suppressors and ameliorates renal aging.

DNA methylation alterations play mechanistic roles in aging; however, the epigenetic regulators/mediators causally involved in renal aging remain elusive. Here, we report that natural and D-galactose (D-gal)-induced aging kidneys display marked suppression of antiaging factor NRF2 (nuclear factor erythroid-derived 2-like 2) and KLOTHO, accompanied by upregulations of DNA methyltransferase (DNMT) 1/3a/3b and NRF2/KLOTHO gene promoter hypermethylations. Administration of a DNMT inhibitor SGI-1072 effectively hypomethylated the promoters, derepressed NRF2/KLOTHO, and mitigated the structural and functional alterations of renal aging in D-gal mice. Moreover, oleuropein (OLP), an olive-derived polyphenol, also displayed similar epigenetic modulation and antiaging effects. OLP inhibited the epigenetic NRF2/KLOTHO suppressions in a gain of DNMT-sensitive manner in cultured renal cells, demonstrating a strong DNA-demethylating capacity. In NRF2 knockout and KLOTHO knockdown D-gal mice, OLP exhibited reduced antiaging effects with KLOTHO displaying a prominent gene effect and effect size; consistently in KLOTHO knockdown mice, the antiaging effects of SGI-1027 were largely abrogated. Therefore, the KLOTHO recovery is critical for the antiaging effects of DNA demethylation. Collectively, our data indicate that aberrant DNMT1/3a/3b elevations and the resultant suppression of antiaging factors contribute significantly to epigenetic renal aging, which might be targeted for epigenetic intervention by synthetic or natural DNA-demethylating agents.

NRF2 is a critical regulator and therapeutic target of metal implant particle-incurred bone damage.

Aseptic metal implant loosening due to wear particle-induced bone damage is a major complication of total joint arthroplasty often leading to revision surgery, of which the key regulators mediating the processes are not clearly defined. Here we reported that in a mouse model of calvarial osteolysis, titanium particles (TiPs) and cobalt-chromium-molybdenum particles induced severe osteolysis accompanied by marked suppression of a master redox transcriptional factor NRF2 (Nuclear factor erythroid derived 2-related factor 2). Nfe2l2 knockout mice treated with TiPs developed worse osteolytic alterations compared with wild-type mice. On the contrary, NRF2 restoration by an NRF2 agonist TBHQ (tert-butylhydroquinone) effectively alleviated the osteolysis and the abnormal expression of NRF2 downstream antioxidant enzymes, inflammatory cytokines and osteogenic factors. Further, TiPs induced adverse osteoblastogenesis and osteoclastogenesis in cultured bone cells, which were substantially blocked by TBHQ in an NRF2 inhibition-sensitive manner. Consistently, the osteoprotective effects of TBHQ observed in wild-type mice were largely limited in Nfe2l2 knockout mice. Collectively, our data suggest that NRF2 suppression is a critical causal event of metal wear particle-incurred osteolysis, and the strategies reinstating NRF2 are effective to lessen the bone damage and potentially reduce the incidence of metal implant loosening.