ADP-ribose transferase PARP16 mediated-unfolded protein response contributes to neuronal cell damage in cerebral ischemia/reperfusion.

Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis, leading to impairment of endoplasmic reticulum (ER) function and activating the unfolded protein response (UPR). PARP16 is an active (ADP-ribosyl)transferase known tail-anchored ER transmembrane protein with a cytosolic catalytic domain. Here, we find PARP16 is highly expressed in ischemic cerebral hemisphere and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated immortalized hippocampal neuronal cell HT22. Using an adeno-associated virus-mediated PARP16 knockdown approach in mice, we find PARP16 knockdown decreases infarct demarcations and has a better neurological outcome after ischemic stroke. Our data indicate PARP16 knockdown decreases ER stress and neuronal death caused by OGD/R, whereas PARP16 overexpression promotes ER stress-mediated cell damage in primary cortical neurons. Furthermore, PARP16 functions mechanistically as ADP-ribosyltransferase to modulate the level of ADP-ribosylation of the corresponding PERK and IRE1α arm of the UPR, and such modifications mediate activation of PERK and IRE1α. Indeed, pharmacological stimulation of the UPR using Brefeldin A partly counteracts PARP16 knockdown-mediated neuronal protection upon OGD/R treatment. In conclusion, PARP16 plays a crucial role in post-ischemic UPR and PARP16 knockdown alleviates brain injury after ischemic stroke. This study demonstrates the potential of the PARP16-PERK/IRE1α axis as a target for neuronal survival in ischemic stroke.

Poly (ADP-ribose) polymerases 16 triggers pathological cardiac hypertrophy via activating IRE1α-sXBP1-GATA4 pathway.

BACKGROUND: Pressure overload-induced pathological cardiac hypertrophy is an independent predecessor of heart failure (HF), which remains the leading cause of worldwide mortality. However, current evidence on the molecular determinants of pathological cardiac hypertrophy is still inadequacy. This study aims to elucidate the role and mechanisms of Poly (ADP-ribose) polymerases 16 (PARP16) in the pathogenesis of pathological cardiac hypertrophy. METHODS: Gain and loss of function approaches were used to demonstrate the effects of genetic overexpression or deletion of PARP16 on cardiomyocyte hypertrophic growth in vitro. Ablation of PARP16 by transducing the myocardium with serotype 9 adeno-associated virus (AAV9)-encoding PARP16 shRNA were then subjected to transverse aortic construction (TAC) to investigate the effect of PARP16 on pathological cardiac hypertrophy in vivo. Co-immunoprecipitation (IP) and western blot assay were used to detect the mechanisms of PARP16 in regulating cardiac hypertrophic development. RESULTS: PARP16 deficiency rescued cardiac dysfunction and ameliorated TAC-induced cardiac hypertrophy and fibrosis in vivo, as well as phenylephrine (PE)-induced cardiomyocyte hypertrophic responses in vitro. Whereas overexpression of PARP16 exacerbated hypertrophic responses including the augmented cardiomyocyte surface area and upregulation of the fetal gene expressions. Mechanistically, PARP16 interacted with IRE1α and ADP-ribosylated IRE1α and then mediated the hypertrophic responses through activating the IRE1α-sXBP1-GATA4 pathway. CONCLUSIONS: Collectively, our results implicated that PARP16 is a contributor to pathological cardiac hypertrophy at least in part via activating the IRE1α-sXBP1-GATA4 pathway, and may be regarded as a new potential target for exploring effective therapeutic interventions of pathological cardiac hypertrophy and heart failure.

The histone methyltransferase DOT1L is a new epigenetic regulator of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with increasing occurrence, high death rates, and unfavorable treatment regimens. The pathogenesis underlying IPF is complex and the epigenetic contributions to IPF are largely unknown. Recent studies have shown that DOT1L (Disruptor of telomeric silencing-1 like), a histone H3K79 methyltransferase, contributes to fibrosis response, but its role in IPF remains unclear. DOT1L, H3K79me3, and the profibrotic proteins levels were upregulated in the pulmonary fibrosis models both in vivo and in vitro. Lentivirus-mediated DOT1L knockdown or DOT1L-specific inhibitor EPZ5676 alleviated the pathogenesis of bleomycin-induced mouse pulmonary fibrosis. Furthermore, heterozygous DOT1L-deficient mice (Dot1l+/-) showed less sensitive to pulmonary fibrosis, as shown by decreased lung fibrosis phenotypes in vivo. Mechanically, DOT1L regulated TGF-ß1-induced fibroblasts fibrosis by increasing enrichments of H3K79me3 on the promoter of Jag1 gene (encoding the Notch ligand Jagged1), enhancing the expression of Jagged1, which in turn stimulated exuberant Notch signaling and actuated the fibrosis response. In conclusion, our study confirmed DOT1L to be an epigenetic modifier in the pathogenesis of lung fibrosis, revealed a counterbalancing mechanism governing Jag1 transcription by modulating H3K79 trimethylation at the Jag1 promoter, activating the Notch signaling, and affecting the expression of profibrotic proteins to accelerate the lung fibrosis.

Histone methyltransferase Smyd2 drives vascular aging by its enhancer-dependent activity.

BACKGROUND: Vascular aging is one of the important factors contributing to the pathogenesis of cardiovascular diseases. However, the systematic epigenetic regulatory mechanisms during vascular aging are still unclear. Histone methyltransferase SET and MYND domain-containing protein 2 (Smyd2) is associated with multiple diseases including cancer and inflammatory diseases, but whether it is involved in endothelial cell senescence and aging-related cardiovascular diseases has not been directly proved. Thus, we aim to address the effects of Smyd2 on regulating angiotensin II (Ang II)-induced vascular endothelial cells (VECs) senescence and its epigenetic mechanism. METHODS AND RESULTS: The regulatory role of Smyd2 in Ang II-induced VECs senescence was confirmed by performing loss and gain function assays. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis was used to systematically screen the potential enhancer during VECs senescence. Here, we found that Smyd2 was significantly upregulated in Ang II-triggered VECs, and deficiency of Smyd2 attenuated senescence-associated phenotypes both in vitro and in vivo. Mechanically, Ang II-induced upregulation of Smyd2 could increase the mono-methylation level of histone 3 lysine 4 (H3K4me1), resulting in a hyper-methylated chromatin state, then further activating enhancers adjacent to key aging-related genes, such as Cdkn1a and Cdkn2a, finally driving the development of vascular aging. CONCLUSIONS: Collectively, our study uncovered that Smyd2 drives a hyper-methylated chromatin state via H3K4me1 and actives the enhancer elements adjacent to key senescence genes such as Cdkn1a and Cdkn2a, and further induces the senescence-related phenotypes. Targeting Smyd2 possibly unveiled a novel therapeutic candidate for vascular aging-related diseases.

Histone methyltransferase Smyd2 drives adipogenesis via regulating STAT3 phosphorylation.

Adipogenesis is a complex cascade involved with the preadipocytes differentiation towards mature adipocytes, accelerating the onset of obesity. Histone methyltransferase SET and MYND domain-containing protein 2 (Smyd2), is involved in a variety of cellular biological functions but the epigenetic regulation of Smyd2 in adipogenesis and adipocyte differentiation remains unclear. Both Smyd2 siRNA and LLY-507, an inhibitor of Smyd2, were used to examine the effect of Smyd2 on adipogenesis and adipocyte differentiation in vitro. Smyd2 heterozygous knockout (Smyd2+/-) mice were also constructed to validate the relationship between Smyd2 and adipogenesis in vivo. We found that Smyd2 is abundant in white adipose tissue and closely correlated with adipocyte differentiation. Knockdown or inhibition of Smyd2 restrained adipocyte differentiation in vitro, which requires the phosphorylation of STAT3. In vivo functional validation, Smyd2+/- mice exert significant fat loss but not susceptible to HFD-induced obesity. Taken together, our findings revealed that Smyd2 is a novel regulator of adipocyte differentiation by regulating the phosphorylation of STAT3, which provides insights into the effects of epigenetic regulation in adipogenesis. Inhibition of Smyd2 might represent a viable strategy for anti-adipogenesis and maybe further alleviate obesity-related diseases in humans.

Inhibition of the cardiac fibroblast-enriched histone methyltransferase Dot1L prevents cardiac fibrosis and cardiac dysfunction.

BACKGROUND: Cardiac fibrosis is characterized by excessive extracellular matrix deposition that contributes to compromised cardiac function and potentially heart failure. Disruptor of telomeric silencing 1-like (Dot1L) is the catalytic enzyme required for histone H3K79 methylation which has been demonstrated to play a role in transcriptional activation. However, the functions of Dot1L in the process of cardiac fibrosis still remain unknown. RESULTS: In the present study, we found that endogenous Dot1L is upregulated in cardiac fibroblasts (CFs) treated with angiotensin II (Ang II) or transforming growth factor (TGF)-ß1, along with elevated extracellular matrix (ECM) such as fibronectin, collagen I and III. Silencing or inhibiting Dot1L mitigated Ang II-induced myofibroblast generation and fibrogenesis. We identified the transcription factor-forkhead box O (FoxO) 3a as a novel substrate of Dot1L, the transcriptional activating mark H3K79me3 level on the promoter of FoxO3a was increase in activated-CFs, and inhibition of Dot1L markedly decreased FoxO3a transcription accompanied by a significant decrease in the expression of fibrogenic gene. Knockdown of FoxO3a could alleviate ECM deposition induced by Ang II, on the contrary, overexpression FoxO3a resulting in CFs activation. Consistently, in vivo Dot1L ablation rescued myocardial ischemia-induced cardiac fibrosis and improved cardiac function. CONCLUSIONS: Our findings conclude that upregulation of Dot1L results in activation of the cardiac fibroblasts to promote profibrotic gene, eventually causes cardiac fibrosis. Pharmacological targeting for Dot1L might represent a promising therapeutic approach for the treatment of human cardiac fibrosis and other fibrotic diseases.

Histone methyltransferase Smyd2 contributes to blood-brain barrier breakdown in stroke.

BACKGROUND: The blood-brain barrier (BBB) plays a principal role in the healthy and diseased central nervous systems, and BBB disruption after ischaemic stroke is responsible for increased mortality. Smyd2, a member of the SMYD-methyltransferase family, plays a vital role in disease by methylation of diverse substrates; however, little is known about its role in the pathophysiology of the brain in response to ischaemia-reperfusion injury. METHODS: Using oxygen glucose deprivation and reoxygenation (OGD/R)-induced primary brain microvascular endothelial cells (BMECs) and Smyd2 knockdown mice subjected to middle cerebral artery occlusion, we evaluated the role of Smyd2 in BBB disruption. We performed loss-of-function and gain-of-function studies to investigate the biological function of Smyd2 in ischaemic stroke. RESULTS: We found that Smyd2 was a critical factor for regulating brain endothelial barrier integrity in ischaemia-reperfusion injury. Smyd2 is upregulated in peri-ischaemic brains, leading to BBB disruption via methylation-mediated Sphk/S1PR. Knockdown of Smyd2 in mice reduces BBB permeability and improves functional recovery. Using OGD/R-induced BMECs, we demonstrated that Sphk/S1PR methylation modification by Smyd2 affects ubiquitin-dependent degradation and protein stability, which may disrupt endothelial integrity. Moreover, overexpression of Smyd2 can damage endothelial integrity through Sphk/S1PR signalling. CONCLUSIONS: Overall, these results reveal a novel role for Smyd2 in BBB disruption in ischaemic stroke, suggesting that Smyd2 may represent a new therapeutic target for ischaemic stroke.

Histone Methyltransferase Dot1L Contributes to RIPK1 Kinase-Dependent Apoptosis in Cerebral Ischemia/Reperfusion.

Background Neuron apoptosis is a pivotal process for brain damage in cerebral ischemia. Dot1L (disruptor of telomeric silencing 1-like) is only known histone H3K79 methyltransferase. It is not clear whether the role and mechanism of Dot1L on cerebral ischemia is related to regulate neuron apoptosis. Methods and Results We use a combination of mice middle cerebral artery occlusion stroke and neurons exposed to oxygen-glucose deprivation followed by reoxygenation to investigate the role and mechanism of Dot1L on cerebral ischemia. We find knockdown or inhibition of Dot1L reversed ischemia-induced neuronal apoptosis and attenuated the neurons injury treated by oxygen-glucose deprivation followed by reoxygenation. Further, blockade of Dot1L prevents RIPK1 (receptor-interacting protein kinase 1)-dependent apoptosis through increased RIPK1 K63-ubiquitylation and decreased formation of RIPK1/Caspase 8 complexes. In line with this, H3K79me3 enrichment in the promoter region of deubiquitin-modifying enzyme A20 and deubiquitinase cylindromatosis gene promotes the increasing expression in oxygen-glucose deprivation followed by reoxygenation -induced neuronal cells, on the contrary, oxygen-glucose deprivation followed by reoxygenation decreases H3K79me3 level in the promoter region of ubiquitin-modifying enzyme cIAP1 (cellular inhibitors of apoptosis proteins), and both these factors ultimately cause K63-deubiquitination of RIPK1. Importantly, knockdown or inhibition of Dot1L in vivo attenuates apoptosis in middle cerebral artery occlusion mice and reduces the extent of middle cerebral artery occlusion -induced brain injury. Conclusions These data support for the first time, to our knowledge, that Dot1L regulating RIPK1 to the apoptotic death trigger contributes to cerebral ischemia injury. Therefore, targeting Dot1L serves as a new therapeutic strategy for ischemia stroke.

SMYD2-mediated TRAF2 methylation promotes the NF-κB signaling pathways in inflammatory diseases.

BACKGROUND: The methylation of lysine residues has been involved in the multiple biological and diseases processes. Recently, some particular non-histone proteins have been elucidated to be methylated by SMYD2, a SET and MYND domain protein with lysine methyltransferase activity. METHODS: SMYD2 was evaluated in synovial tissue and cells derived from rheumatoid arthritis patients. We confirmed TRAF2 could be methylated by SMYD2 using Mass spectrometry, pull-down, immunoprecipitation, methyltransferase assay, ubiquitination assay, luciferase reporter assays, and western blot analyses. Using loss- and gain-of function studies, we explored the biological functions of SMYD2 in vitro and in vivo. Using acute and chronic inflammation with different mice models to determine the impact of SMYD2. RESULTS: Here, we first time confirmed that the cytoplasmic protein TRAF2 as the kernel node for NF-κB signaling pathway could be methylated by SMYD2. SMYD2-mediated TRAF2 methylation contributed to the durative sensitization of NF-κB signaling transduction through restraining its own proteolysis and enhancing the activity. In addition, we found knocking down of SMYD2 has different degrees of mitigation in acute and chronic inflammation mice models. Furthermore, as the lysine-specific demethylase, LSD1 could resist methylation on TRAF2 induced by SMYD2. CONCLUSIONS: Our data uncovered an unprecedented cytoplasmic protein network that employed methylation of TRAF2 for the maintenance of NF-κB activation during inflammatory diseases.

Neogenkwanine I from the flower buds of Daphne genkwa with its stereostructure confirmation using quantum calculation profiles and antitumor evaluation.

Neogenkwanine I (1), a new daphnane-type diterpene with 4,7-ether group, along with four known ones (2-5), were isolated from Daphne genkwa. The structure including absolute configurations of 1 was established on the basis of NMR, 13C-NMR and ECD calculations and CD exciton chirality analysis. 13C-NMR and ECD calculations of daphnane-type diterpenes were reported here for the first time. All of the diterpenes were screened for their cytotoxic activities against MCF-7 and Hep3B cell lines. The cytotoxicity structure- activity relationship of compounds was illustrated with the absence of ortho-ester group of daphnane-type diterpenes.