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Dysregulation of microRNAs has been implicated in diverse diseases, including Alzheimer's disease (AD). MiR-191-5p in plasma/serum has been identified as a novel and promising noninvasive diagnostic biomarker for AD. However, whether miR-191-5p is involved in AD pathogenesis is largely unknown, and its levels in human AD brains are undetermined. Herein, we demonstrated that miR-191-5p downregulated tau phosphorylation at multiple AD-related sites and promoted neurite outgrowth using immunoblotting, immunofluorescence, and neurite outgrowth assays. Moreover, immunoblotting and enzyme-linked immunosorbent assays indicated that miR-191-5p decreased amyloid precursor protein phosphorylation levels and beta-amyloid (Aβ) generation. Furthermore, miR-191-5p reduced ceramide-induced neuronal cell death analyzed by trypan blue staining, the in situ cell death detection kit, and Annexin V-FITC/PI flow cytometry. Next, we verified that death-associated protein kinase 1 (DAPK1) was a direct target of miR-191-5p through the dual luciferase reporter assay and confirmed that the effects of miR-191-5p were antagonized by restoration of DAPK1 expression. Finally, the hippocampal miR-191-5p level was found to be decreased in humans with AD compared with controls and was inversely correlated with the DAPK1 expression level. Collectively, these findings suggest that miR-191-5p might exert inhibitory effects on tau phosphorylation, Aβ secretion, and neuronal cell death by directly targeting DAPK1, providing an attractive therapeutic option for AD.

Objective: To reveal the possible effects of death-associated protein kinase 1 (DAPK1) on the progression of osteoarthritis (OA) and the potential underlying mechanism. Methods: The expression of DAPK1 in OA and normal samples and interleukin (IL)-1β-stimulated chondrocytes was analyzed by quantitative real-time polymerase chain reaction and Immunoblot assay. Cell viability, proliferation, and apoptosis in DAPK1-knockdown cells stimulated with IL-1β were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution, 5-ethynyl-2′-deoxyuridine staining and flow cytometry. The chondrocyte degradation and inflammatory response in IL-1β-induced chondrocytes were investigated by Immunoblot analysis and enzyme-linked-immunosorbent serologic assay. In addition, the effect of DAPK1 on p38 mitogen-activated protein kinase (MAPK) activation was analyzed by immunoblot assay. Results: This study revealed that DAPK1 was highly expressed in OA patients and IL-1β-induced chondrocytes. Down-regulation of DAPK1 enhanced IL-1β-induced chondrocyte proliferation. DAPK1 knockdown inhibited IL-1β-induced chondrocyte degradation. In addition, DAPK1 depletion inhibited IL-1β-induced chondrocyte inflammation. Mechanically, it was revealed that down-regulation of DAPK1 could inhibit the p38 MAPK pathway, and therefore affected progression of OA. Conclusion: DAPK1 knockdown attenuates IL-1β-induced extracellular matrix degradation and inflammatory response in OA chondrocytes by regulating the p38 MAPK pathway.

Human immunodeficiency virus-1 (HIV-1) infection leads to the development of acquired immunodeficiency syndrome (AIDS). To establish a productive infection, HIV-1 hijacks the cellular machinery and modulates various physiological processes to propagate itself. The pathways altered by HIV-1 include cell cycle, autophagy, apoptosis, cell stress pathways, immune response, antiviral response, etc. Zipper interacting protein kinase (ZIPK) is a member of the death-associated protein kinase (DAPK) family of proteins, known to be one of the key regulators of cell death and cell survival pathways. ZIPK is also involved in regulating many cellular processes that are altered during HIV-1 infection; thus, we have explored the functional role of ZIPK in HIV-1 infection. Our results show that ZIPK protein expression is downregulated during HIV-1 infection in Nef dependent manner. Overexpression of ZIPK leads to downregulation in LTR-driven gene expression and virus production, whereas ZIPK knockdown induces viral gene expression and replication. HIV-1 promoter activity is reportedly enhanced by Nef-mediated activation of some transcription factors like NFκB and STAT3. ZIPK is reported to inhibit the STAT3 activity by phosphorylating it at ser-727. Our results show that STAT3 (ser-727) phosphorylation is decreased upon overexpression of Nef with simultaneous downregulation of ZIPK expression. We finally show that HIV-1 Nef interacts with ZIPK and induces its proteasomal degradation. Overall, our data suggests that Nef is involved in downregulation of ZIPK thereby increasing the virus production through rescue of STAT3 activity.

Diabetic nephropathy (DN) is one of the most serious complications of advanced diabetes, and increases patient mortality. Recently, epigenetics-mediated hyperglycemic memory in pathological process of DN has received attention. The purpose of this study was to determine the underlying mechanism by which sirt7 modulates hyperglycemic memory in DN. In glomerular endothelial cells (GECs) cultured in high glucose and glomeruli of DN patients and rats, an increase in p65 phosphorylation and endothelial adhesion molecule levels persisted after glucose normalization but was reversed by glucose normalization associated with death-associated protein kinase-3 (DAPK3) knockout or DAPK3 inhibitor. High glucose-mediated decrease in sirt7, the deacetylase modulating H3K18-acetylation (H3K18ac), was sustained after normoglycemia. Sirt7 overexpression accompanied by glucose normalization suppressed DAPK3 expression and inflammation in GECs. Moreover, sh-sirt7-induced inflammation was inhibited by si-DAPK3. Furthermore, sirt7 and H3K18ac were located at the DAPK3 promoter region. ELK1 was found to combine with sirt7. si-ELK1 supplemented with normoglycemia inhibited high glucose-induced DAPK3 expression and inflammation in GECs. ELK1 overexpression-mediated inflammation was inhibited by si-DAPK3. In addition, ELK1 and sirt7 were located at the same promoter region of DAPK3. ELK1 overexpression enhanced DAPK3 promoter activity, which disappeared after specific binding site mutation. In vivo, sirt7 overexpression decreased inflammation and improved renal function during insulin treatment of DN rats, whereas insulin alone did not work. Our data demonstrated high glucose-mediated mutual inhibition between sirt7 and ELK1 induced DAPK3 transcription and inflammation despite normoglycemia in GECs, thus forming a vicious cycle and participating in the occurrence of hyperglycemic memory in DN.

The autophagy pathway is used by eukaryotic cells to maintain metabolic homeostasis. Autophagy has two functions in cancerous cells which could inhibit tumorigenesis or lead to cancer progression by increasing cell survival and proliferation.In this review article, Web of Science, PubMed, Scopus, and Google Scholar were searched and summarized published studies to explore the relationship between DAPK1 and mTORC1 signaling association on autophagy in cancer. Autophagy is managed through various proteins including the mTOR, which is two separated structural and functional complexes known as mTORC1 and mTORC2. MTORC1 is an important component of the regulatory pathway affecting numerous cellular functions including proliferation, migration, invasion, and survival. This protein plays a key role in human cancers. The activity level of mTORC1 is regulated by the death-associated protein kinases (DAPks) family, especially DAPK1. In many cancers, DAPK1 acts as a tumor suppressor which can be attributed to its ability to suppress cellular transformation and to inhibit metastasis.A deep investigation not only will reveal more about the function of DAPK1 but also might provide insights into novel therapies aimed to modulate the autophagy pathway in cancer and to achieve better cancer therapy.

Objective. Heart failure and ventricular remodeling after acute myocardial infarction (AMI) are important factors affecting the prognosis of patients. Therefore, we expected to explore the therapeutic target of AMI by studying the effect of death-associated protein kinase 1 (DAPK1) on AMI rat model. Materials and Methods. We used male Sprague-Dawley rats to make AMI model, and after 1, 3, 7, and 14 d, we detect the success rate of modeling and the expression change of DAPK1 through 2, 3, 5-triphenyl tetrazolium chloride staining, myocardial injury markers detection, echocardiographic detection, and histological experiment. In addition, we determined the effect of DAPK1 on AMI by subcutaneous injection of the DAPK1 inhibitor (TC-DAPK 6). The effect of DAPK1 on cardiomyocytes has also been verified in cell experiments on H9c2 cells. Results. The expression of DAPK1 in AMI rats was significantly higher than that in sham group, and it increased with time. The expression of inflammatory factors (interleukin- (IL-) 1β, IL-6, and tumor necrosis factor-α) in AMI rats treated by TC-DAPK 6 was reduced. In addition, TC-DAPK 6 also reduced the activity of malonaldehyde and increased the activities of superoxide dismutase, glutathione, and catalase. The expression of antioxidant molecules such as peroxiredoxin1/4 and glutathione peroxidase1/3 was also promoted by TC-DAPK 6. In H9c2 cells, TC-DAPK 6 also reduced its oxidative stress level. Conclusions. The increase of DAPK1 may be related to the pathogenesis of AMI. DAPK1 inhibitors protect cardiomyocytes from AMI-induced myocardial injury by reducing levels of inflammation and oxidative stress in myocardial tissue and cells.

Background Death-associated protein kinase (DAPK) has a strong function of tumor suppression involving apoptosis regulation, autophagy, and metastasis inhibition. Hypermethylation of CpG islands in DAPK gene promoter region is one of the important ways to inactivate this tumor suppressor gene, which might promote lung carcinogenesis. However, the clinicopathological significance of the DAPK promoter hypermethylation in lung cancer remains unclear. In this study, we performed a meta-analysis trying to estimate the clinicopathological significance of DAPK promoter hypermethylation in non-small cell lung cancer (NSCLC). Methods A detailed literature search for publications relevant to DAPK gene promoter methylation and NSCLC was made in PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure, CSTJ, Wanfang databases, and SinoMed (CBM). The random-effects model and fixed-effects model were utilized to pool the relative ratio based on the heterogeneity test in the meta-analysis. Results A total of 41 studies with 3348 patients were included. The frequency of DAPK methylation was significantly higher in NSCLC than in non-malignant control (odds ratio (OR) = 6.88, 95% confidence interval (CI): 4.17–11.35, P Conclusion DAPK promoter hypermethylation might be a candidate diagnostic and prognostic tumor marker for NSCLC.

Prostate cancer (PCa) is a malignant tumor that originates in the male genitourinary system. Downregulation of death-associated protein kinase 1 (DAPK1) is closely related to PCa. Little is known about the functional role of DAPK1 in regulating cancer stem cell (CSC)-like characteristics of PCa cells, and we have conducted research on this topic. Compared with tumor-adjacent normal tissues, DAPK1 was severely downregulated in tumor tissues of PCa patients. DAPK1 expression was also reduced in PCa cell lines with respect to that in normal prostate cells. Moreover, we sorted PCa-CSCs (PCa-CD133

Gastric cancer is one of the most common malignant cancers globally. Chemotherapy resistance remains a major obstacle in the treatment of gastric cancer, and the molecular mechanisms underlying drug resistance are still not well understood. We previously reported that Zipper interacting protein kinase (ZIPK), also known as death‐associated protein kinase3, exerts an oncogenic effect on gastric cancer via activation of Akt/NF‐κB signaling and promotion of stemness. Here, we explored the roles of ZIPK in cisplatin resistance. We report that ZIPK enhances cell proliferation and invasion and reduces the antitumor activity of cisplatin in gastric cancer. In addition, our western blot data suggest that ZIPK activated the IL‐6/STAT3 signaling pathway. Furthermore, ZIPK increased the expression of IL‐6 and multidrug‐resistance genes. Using the STAT3 inhibitor stattic to block the IL‐6/STAT3 signaling pathway strongly increased the sensitivity of ZIPK‐expressed cells to cisplatin. In conclusion, ZIPK may play a role in cisplatin resistance through activation of the IL‐6/ STAT3 signaling pathway. Inhibition of STAT3 in gastric cancer overexpressing ZIPK might have potential to improve the efficacy of cisplatin., ZIPK activates the IL‐6/STAT3 signaling pathway via Ser727 phosphorylation in gastric cancer cells and increases IL‐6‐induced STAT3‐dependent transcription. Effective blockade of STAT3 activity by the STAT3 inhibitor stattic sensitized gastric cancer cells to cisplatin and reduced expression of the cisplatin resistance‐related genes G3BP2, THOC1, ATP7A, and OTUD1.

Death-associated protein kinase 1 (DAPK1), as a calcium/calmodulin (CaM) regulated serine/threonine kinase, functions in apoptotic and autophagy pathways and represents an interesting drug target for inflammatory bowel disease and Alzheimer’s disease. The crystal structure of the DAPK1 catalytic domain and the autoregulatory domain (ARD) in complex with CaM provides an understanding of CaM-dependent regulation of DAPK1 activity. However, the molecular basis of how distinct Trp305 (W305Y and W305D) mutations in the ARD modulate different DAPK1 activities remains unknown. Here, we performed multiple, μs-length molecular dynamics (MD) simulations of the DAPK1–CaM complex in three different (wild-type, W305Y, and W305D) states. MD simulations showed that the overall structural complex did not change significantly in the wild-type and W305Y systems, but underwent obvious conformational alteration in the W305D system. Dynamical cross-correlation and principal component analyses revealed that the W305D mutation enhanced the anti-correlated motions between the DAPK1 and CaM and sampled a broader distribution of conformational space relative to the wild-type and W305Y systems. Structural and energetical analyses further exhibited that CaM binding was unfavored in response to the W305D mutation, resulting in the decreased binding of CaM to the W305D mutant. Furthermore, the hydrogen bonds and salt bridges responsible for the loss of CaM binding on the interface of the DAPK1–CaM complex were identified in the W305D mutant. This result may provide insights into the key role of Trp305 in the regulation of CaM-mediated DAPK1 activity.

Background Ewing sarcoma (ES) is the second most common primary malignant bone tumor mainly occurring in children, adolescents and young adults with high metastasis and mortality. Autophagy has been reported to be involved in the survival of ES, but the role remains unclear. Therefore, it’s necessary to investigate the prognostic value of autophagy related genes using bioinformatics methods. Results ATG2B, ATG10 and DAPK1 were final screened genes for a prognostic model. KM and risk score plots showed patients in high score group had better prognoses both in training and validation sets. C-indexes of the model for training and validation sets were 0.68 and 0.71, respectively. Calibration analyses indicated the model had high prediction accuracy in training and validation sets. The AUC values of ROC for 1-, 3-, 5-year prediction were 0.65, 0.73 and 0.84 in training set, 0.88, 0.73 and 0.79 in validation set, which suggested high prediction accuracy of the model. Decision curve analyses showed that patients could benefit much from the model. Differential and functional analyses suggested that autophagy and apoptosis were upregulated in high risk score group. Conclusions ATG2B, ATG10 and DAPK1 were autophagy related genes with potential protective function in ES. The prognostic model established by them exhibited excellent prediction accuracy and discriminatory capacities. They might be used as potential prognostic biomarkers and therapeutic targets in ES.

Death-associated protein kinase 3 (DAPK3) is a serine/threonine protein kinase that regulates apoptosis, autophagy, transcription, and actin cytoskeleton reorganization. DAPK3 induces morphological alterations in apoptosis when overexpressed, and it is considered a potential drug target in antihypertensive and anticancer drug development. In this article, we report new findings from a structure-guided virtual screening for discovery of phytochemicals that could modulate the elevated expression of DAPK3, and with an eye to anticancer drug discovery. We used the Indian Medicinal Plants, Phytochemistry and Therapeutics (IMPPAT), a curated database, as part of the methodology. The potential initial hits were identified based on their physicochemical properties and binding affinity toward DAPK3. Subsequently, various filters for drug likeness followed by interaction analysis and molecular dynamics (MD) simulations for 100 nsec were performed to explore the conformational sampling and stability of DAPK3 with the candidate molecules. Notably, the data from all-atom MD simulations and principal component analysis suggested that DAPK3 forms stable complexes with ketanserin and rotenone. In conclusion, this study supports the idea that ketanserin and rotenone bind to DAPK3, and show stability, which can be further explored as promising scaffolds in drug development and therapeutics innovation in clinical contexts such as hypertension and various types of cancer.

The aberrant methylation of many genes has been reported to be associated with various carcinomas. Accurate detection of the methylation level could provide critical insights into the diagnostic analysis of diseases. Here, a sensitive HpaII-edited absolute droplet loop-mediated isothermal amplification (HEADLAMP) method based on methylation-sensitive restriction enzyme (MSRE) HpaII was developed for the digital quantification of DNA methylation. Methylation levels of the death-associated protein kinase 1 (DAPK1) gene that is associated with many cancers were studied using β-actin as an internal reference. DAPK1 (2.5 pM) with 0.01% methylation (250 aM) can be detected with the conventional HpaII-edited LAMP assay. Using HEADLAMP, as low as 1% methylation level can be distinguished with an estimated limit of detection of 5 aM (ca. 3 copies/μL). Moreover, HEADLAMP can detect low levels of methylated DAPK1 in normal L-02 cells, while the conventional assay cannot. Finally, HEADLAMP was applied to the detection of DAPK1 methylation in 20 clinical tissue samples, which revealed hypermethylated DAPK1 in cervical cancer patients. We envisage potential applications of this robust, specific, and sensitive HEADLAMP assay in epigenetic studies and early clinical diagnosis.

Emerging evidence has suggested that miRNAs are important regulators of intestinal I/R injury, but their function in this context remains elusive. To evaluate the role of miR-26b-5p in intestinal I/R injury. We utilized in vivo murine models of intestinal I/R and in vitro Mode-K cell-based models of oxygen and glucose deprivation/reperfusion (OGD/R) to examine the function of miR-26b-5p in intestinal I/R injury. The expression of miR-26b-5p in intestinal mucosa and Mode-K cell was detected by RT-PCR. HE staining and Chiu’s score were used to evaluate intestinal mucosa injury severity. Apoptosis was detected by TUNEL stain, flow cytometry, and western blot. TargetScan and StarBase prediction algorithms were applied to predict putative target genes of miR-26b-5p and validated by luciferase reporter analyses. We found that the expression of miR-26b-5p in intestinal mucosa was markedly decreased during I/R injury. We additionally found miR-26b-5p overexpression to markedly disrupt intestinal I/R- or OGD/R-induced injury in vivo and in vitro, whereas inhibiting this miRNA had an adverse impact and resulted in increased intestinal tissue injury and Mode-K cell damage. From a mechanistic perspective, miR-26b-5p was predicted to target DAPK1, which was related to cellular apoptosis. Luciferase reporter assay results confirmed that miR-26b-5p directly targets DAPK1 in Mode-K cells, thereby suppressing OGD/R-induced cell apoptosis. Our findings show that miR-26b-5p may prevent intestinal I/R injury via targeting DAPK1 and inhibiting intestinal mucosal cell apoptosis, suggesting that this miRNA may be a viable target for the treatment of intestinal I/R injury.

Sharma and colleagues identify the kinase DAPK3 as a positive regulator of the STING-interferon-beta activation pathway. DAPK3 acts to modify E3 ubiquitin ligases that regulate STING K63-linked poly-ubiquitination. Evasion of host immunity is a hallmark of cancer; however, mechanisms linking oncogenic mutations and immune escape are incompletely understood. Through loss-of-function screening of 1,001 tumor suppressor genes, we identified death-associated protein kinase 3 (DAPK3) as a previously unrecognized driver of anti-tumor immunity through the stimulator of interferon genes (STING) pathway of cytosolic DNA sensing. Loss of DAPK3 expression or kinase activity impaired STING activation and interferon (IFN)-beta-stimulated gene induction. DAPK3 deficiency in IFN-beta-producing tumors drove rapid growth and reduced infiltration of CD103(+)CD8 alpha(+) dendritic cells and cytotoxic lymphocytes, attenuating the response to cancer chemo-immunotherapy. Mechanistically, DAPK3 coordinated post-translational modification of STING. In unstimulated cells, DAPK3 inhibited STING K48-linked poly-ubiquitination and proteasome-mediated degradation. After cGAMP stimulation, DAPK3 was required for STING K63-linked poly-ubiquitination and STING-TANK-binding kinase 1 interaction. Comprehensive phospho-proteomics uncovered a DAPK3-specific phospho-site on the E3 ligase LMO7, critical for LMO7-STING interaction and STING K63-linked poly-ubiquitination. Thus, DAPK3 is an essential kinase for STING activation that drives tumor-intrinsic innate immunity and tumor immune surveillance.

Leucocyte adhesion to the vascular endothelium is a critical event in the early inflammatory response to infection and injury. This process is primarily regulated by the expression of cell adhesion molecules (CAMs) in endothelial cells. It has been well documented that tumor necrosis factor alpha (TNF-α) is a key regulator of CAM expression within this process, but its regulatory mechanism remains controversial. To investigate the scenario within this process, we assessed the role of zipper-interacting protein kinase (ZIPK), a serine/threonine kinase with multiple substrates, in CAM expression. We used TNF-α as inflammatory stimulator and found that ZIPK was integrated into the signaling regulation of TNF-α-mediated CAM expression. In human umbilical vein endothelial cells (HUVECs), TNF-α exposure led to significantly increased expression of both intercellular CAM-1 (ICAM-1) and vascular CAM-1 (VCAM-1), along with an increase in the adhesion of THP-1 monocytes to HUVECs. Simultaneously, ZIPK gene was also up-regulated at the transcription level. These effects were clearly inhibited by the ZIPK-specific inhibitor Tc-DAPK6 or small interfering RNA (siRNA) capable of specifically inhibiting ZIPK expression. We thus suggest that both ZIPK activation and ZIPK gene expression are necessary for TNF-α-mediated CAM expression and leucocyte adhesion. Interestingly, ZIPK inhibition also significantly suppressed TNF-α-induced nuclear factor kappa B (NF-κB) activation, indicating that TNF-α-mediated ZIPK expression functions upstream of NF-κB and CAM expression. We thus propose a TNF-α/ZIPK/NF-κB signaling axis for CAM expression that is necessary for leucocyte adhesion to endothelial cells. Our data in this study revealed a potential molecular target for exploring anti-inflammation drugs.

Perinatal hypoxic ischemia encephalopathy (HIE) is a serious disease occurring in neonate. Growing studies have already validated the pivotal function of microRNAs (miRNAs) in a variety of diseases. However, whether miR-130a-3p participated in neonatal HIE remains vague. In this study, we planned to explore the molecular mechanism of H19/miR-130a-3p/DAPK1 axis in HIE. We established a in vivo mice model induced by middle cerebral artery occlusion/reperfusion (MCAO/R) and in vitro models of SH-SY5Y and N2a cells following oxygen-glucose deprivation and reperfusion (OGD/R) treatment. DAPK1 is widely explored in multiple diseases and bioinformatic analysis indicated miR-130a-3p potentially targeted DAPK1. We found DAPK1 expression was upregulated while miR-130a-3p expression was downregulated in HIE, MCAO/R mice model and OGD/R treated SH-SY5Y and N2a cells. Moreover, miR-130a-3p was verified to target DAPK1. DAPK1 upregulation restored the inhibitory effect of miR-130a-3p elevation on SH-SY5Y and N2a cells apoptosis as well as on cerebral damage by I/R. In addition, H19 was confirmed to bind with miR-130a-3p in SH-SY5Y and N2a cells. H19 and miR-130a-3p coordinately regulated SH-SY5Y and N2a cells apoptosis as well as cerebral damage by I/R. In conclusion, H19/miR-130a-3p/DAPK1 axis regulated the pathophysiology of neonatal HIE, suggesting potential therapeutic targets for neonatal HIE treatment.

Epilepsy is a chronic encephalopathy and one of the most common neurological disorders. Death-associated protein kinase 1 (DAPK1) expression has been shown to be upregulated in the brains of human epilepsy patients compared with those of normal subjects. However, little is known about the impact of DAPK1 on epileptic seizure conditions. In this study, we aim to clarify whether and how DAPK1 is regulated in epilepsy and whether targeting DAPK1 expression or activity has a protective effect against epilepsy using seizure animal models. Here, we found that cortical and hippocampal DAPK1 activity but not DAPK1 expression was increased immediately after convulsive pentylenetetrazol (PTZ) exposure in mice. However, DAPK1 overexpression was found after chronic low-dose PTZ insults during the kindling paradigm. The suppression of DAPK1 expression by genetic knockout significantly reduced PTZ-induced seizure phenotypes and the development of kindled seizures. Moreover, pharmacological inhibition of DAPK1 activity exerted rapid antiepileptic effects in both acute and chronic epilepsy mouse models. Mechanistically, PTZ stimulated the phosphorylation of NR2B through DAPK1 activation. Combined together, these results suggest that DAPK1 regulation is a novel mechanism for the control of both acute and chronic epilepsy and provide new therapeutic strategies for the treatment of human epilepsy.

Death-associated protein kinase 3 (DAPK3), a member of the DAPK family, contributes to cytokinesis by phosphorylating myosin II regulatory light chain (MRLC). Missense mutations in DAPK3, T112M, D161N, and P216S, were observed in the lung, colon, and cervical cancers, respectively, but the effects of these mutations on cytokinesis remain unclear. Here, we show that cells expressing EGFP-DAPK3-T112M, -D161N, or -P216S exhibited reduced rates of cytokinesis, with an increased ratio of multinucleated cells. In addition, these cells exhibited reduced levels of phosphorylated MRLC at the contractile ring. Collectively, our data demonstrates that cancer-associated DAPK3 mutations impair cytokinesis by reducing phosphorylated MRLC.

Highlights • The kinase Dapk2 is quickly induced in activated T cells. • Dapk2 deficient T cells promote GC responses by increasing cognate T-B interactions. • mTORC1 pathway mediates Dapk2 function in T cell., Germinal center (GC) formation is a critical step during T-dependent humoral immune responses. We report Death Associated Protein Kinase 2, a serine/threonine kinase, is rapidly induced in T cells following activation and plays an inhibitory role in T cell-mediated help for the GC formation. Specifically, T cells deficient in Dapk2 have an increased ability to physically conjugate with antigen-presenting B cells and to promote GC formation. However, Dapk2 does not regulate T cell receptor signaling strength and does not influence cytokine-driven T-cell subset polarization. Instead, Dapk2 dampens mTORC1 activities by associating with Raptor. Silencing of Raptor rescues defects observed with the Dapk2 insufficiency. Our study thus identifies Dapk2 as a new kinase likely involved in negative regulation of contact-dependent help delivery to B cells and GC formation.

Serine/threonine kinase 17A (death-associated protein kinase-related apoptosis-inducing protein kinase 1─DRAK1) is a part of the death-associated protein kinase (DAPK) family and belongs to the so-called dark kinome. Thus, the current state of knowledge of the cellular function of DRAK1 and its involvement in pathophysiological processes is very limited. Recently, DRAK1 has been implicated in tumorigenesis of glioblastoma multiforme (GBM) and other cancers, but no selective inhibitors of DRAK1 are available yet. To this end, we optimized a pyrazolo[1,5

BackgroundPolycystic ovary syndrome (PCOS), the most common heterogeneous reproductive disease afflicting women of childbearing age, has been recognized as a chronic inflammatory disease recently. Most PCOS patients have hyperandrogenism, indicating a poor prognosis and poor pregnancy outcomes. The molecular mechanism underlying PCOS development is still unknown. In the present study, we investigated the gene expression profiling characteristics of PCOS with hyperandrogenism (HA) or without hyperandrogenism (NHA) and identified immune-related factors that correlated with embryo implantation failure.MethodsPCOS and recurrent implantation failure (RIF) microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. ClueGO software was used to perform enrichment analysis of differentially expressed genes (DEGs) in PCOS with varying androgen levels. The Weighted Co-Expression Network Analysis (WGCNA) was used to identify co-expressed modules and shared gene signatures between HA PCOS and RIF. Moreover, the upregulated DEGs of HA PCOS and RIF were intersected with shared gene signatures screening by WGCNA to excavate further key prognostic biomarkers related to implantation failure of HA PCOS. The selected biomarker was verified by qRT-PCR.ResultsA total of 271 DEGs were found in HA PCOS granulosa cell samples, and 720 DEGs were found in NHA PCOS. According to CuleGO enrichment analysis, DEGs in HA PCOS are enriched in immune activation and inflammatory response. In contrast, DEGs in NHA PCOS are enriched in mesenchymal cell development and extracellular space. Using WGCNA analysis, we discovered 26 shared gene signatures between HA PCOS and RIF, which were involved in corticosteroid metabolism, bone maturation and immune regulation. DAPK2 was furtherly screened out and verified to be closely related with the development of HA PCOS, acting as an independent predictor biomarker of the embryo implantation failure. DAPK2 expression was negatively correlated to the embryo implantation rate (r=-0.474, P=0.003). The immune infiltration results suggested that upregulated DAPK2 expression was closely related with NK cell infiltration and macrophage M2, playing an essential role in the pathogenesis of implantation failure in HA PCOS.ConclusionOur research revealed the expression profiling of PCOS with different androgen levels and identified DAPK2 as a critical prognostic biomarker for implantation failure in PCOS.

Mdivi-1 is widely used as a pharmacological tool to inhibit dynamin-related protein-1-mediated mitochondrial fission. Whether this compound may interact directly or indirectly with ion channels or cellular pathways fundamental for the regulation of vascular smooth muscle tone remains unknown. The present study aimed to assess the effect of mdivi-1 on Ca

Glutamate excitotoxicity induces neuronal cell death during epileptic seizures. Death-associated protein kinase 1 (DAPK1) expression is highly increased in the brains of epilepsy patients; however, the underlying mechanisms by which DAPK1 influences neuronal injury and its therapeutic effect on glutamate excitotoxicity have not been determined. We assessed multiple electroencephalograms and seizure grades and performed biochemical and cell death analyses with cellular and animal models. We applied small molecules and peptides and knocked out and mutated genes to evaluate the therapeutic efficacy of kainic acid (KA), an analog of glutamate-induced neuronal damage. KA administration increased DAPK1 activity by promoting its phosphorylation by activated extracellular signal-regulated kinase (ERK). DAPK1 activation increased seizure severity and neuronal cell death in mice. Selective ERK antagonist treatment, DAPK1 gene ablation, and uncoupling of DAPK1 and ERK peptides led to potent anti-seizure and anti-apoptotic effects in vitro and in vivo. Moreover, a DAPK1 phosphorylation-deficient mutant alleviated glutamate-induced neuronal apoptosis. These results provide novel insight into the pathogenesis of epilepsy and indicate that targeting DAPK1 may be a potential therapeutic strategy for treating epilepsy.

Renal cell carcinoma (RCC) is a kind of malignant tumor originating from the renal tubular epithelium. Approximately 30% of patients with renal cancer are found to have metastasis when first diagnosed. Exploring other effective treatment methods in addition to surgery is an urgent need in the research field of renal cell carcinoma. Polybromo 1 (PBRM1) is the second most mutated gene in RCC, with a mutation rate of ~40%. Notably, the posttranscriptional modification of PBRM1 in RCC is unclear. In this study, we performed unbiased mass spectrometry of PBRM1 and identified ubiquitin-protein ligase E3A (UBE3A), an extensively studied E3 ligase that can bind with PBRM1 and regulate the stability of PBRM1 in renal cancer cells. We further found that RBPJ/DAPK3 modulated the E3 ligase activity of UBE3A by interfering with the PKA phosphorylation of UBE3A. Finally, we demonstrated that the RBPJ/DAPK3/UBE3A/PBRM1/p21 axis contributed to the sensitivity of renal cancer cells to CDK4/6 inhibitors. In addition, in combination with RBPJ inhibitors, CDK4/6 inhibitors showed synergistically enhanced effects on renal cancer cells. In summary, we not only revealed a novel RBPJ/DAPK3/UBE3A/PBRM1/p21 signaling axis but also identified a combination strategy for overcoming the resistance of renal cancer cells to CDK4/6 inhibitors.

Background Extracellular vesicles (EVs) derived from tumor-associated macrophages are implicated in the progression and metastasis of gastric cancer (GC) via the transfer of molecular cargo RNAs. We aimed to decipher the impact of microRNA (miR)-15b-5p transferred by M2 macrophage-derived EVs in the metastasis of GC. Methods Expression of miR-15b-5p was assessed and the downstream genes of miR-15b-5p were analyzed. GC cells were subjected to gain- and loss-of function experiments for miR-15b-5p, BRMS1, and DAPK1. M2 macrophage-derived EVs were extracted, identified, and subjected to co-culture with GC cells and their biological behaviors were analyzed. A lung metastasis model in nude mice was established to determine the effects of miR-15b-5p on tumor metastasis in vivo. Results miR-15b-5p was upregulated in GC tissues and cells as well as in M2 macrophage-derived EVs. miR-15b-5p promoted the proliferative and invasive potentials, and epithelial-mesenchymal transition (EMT) of GC cells. M2 macrophage-derived EVs could transfer miR-15b-5p into GC cells where it targeted BRMS1 by binding to its 3’UTR. BRMS1 was enriched in the DAPK1 promoter region and promoted its transcription, thereby arresting the proliferative and invasive potentials, and EMT of GC cells. In vivo experiments demonstrated that orthotopic implantation of miR-15b-5p overexpressing GC cells in nude mice displayed led to enhanced tumor metastasis by inhibiting the BRMS1/DAPK1 axis. Conclusions Overall, miR-15b-5p delivered by M2 macrophage-derived EVs constitutes a molecular mechanism implicated in the metastasis of GC, and may thus be considered as a novel therapeutic target for its treatment.

Signal transducer and activator of transcription 3, STAT3, is an essential member of the STAT family. STAT3 regulates diverse genes mediating inflammatory reaction, cell survival, proliferation, and angiogenesis, and it is aberrantly upregulated and activated in various types of malignancies. Meanwhile, STAT3 signaling is involved in multiple feedback loops and pathways. In this study, we demonstrate that in renal cell carcinoma, miR-93-3p act an oncogenesis role in renal cell carcinoma. It enhanced RCC cell proliferation and suppressed apoptosis. Besides, STAT3 could regulate the transcription of miR-93 by directly binding with its promoter region. miR-93 can inhibit the protein level of death-associated protein kinase 1, DAPK1. What’s more, STAT3 could block the expression of DAPK1 on the level of RNA. Importantly, we verify that, through over-expression, DAPK1 might, in return, suppress the activated-STAT3 entering cell nucleus. Thus, the study uncovers a potential signaling transduction pathway, STAT3-miR93-DAPK1, which is continuously activated, and may provide a novel clinical therapeutic approach for RCC.

We investigated the prognostic significance of Death-Associated Protein Kinase 1 (DAPK1) and its role in sunitinib resistance in clear cell renal cell carcinoma (ccRCC). DAPK1 mRNA levels were significantly lower in tumor tissues than normal kidney tissues in TCGA-KIRC dataset (n=428). Both overall survival and disease-free survival were significantly shorter in ccRCC patients with low DAPK1 expression than those with high DAPK1 expression. Receiver operating characteristic curve analysis showed that low DAPK1 expression correlated with poor prognosis in ccRCC patients. Multivariate analysis confirmed that DAPK1 expression was an independent prognostic indicator in ccRCC. Gene set enrichment analysis showed that low DAPK1 expression correlates with upregulation of pathways related to metastasis, drug resistance, hypoxia and invasiveness in ccRCC patients. Sunitinib-resistant ccRCC cells show significantly lower DAPK1 mRNA and protein levels than sunitinib-sensitive ccRCC cells. DAPK1 overexpression enhances apoptosis in sunitinib-resistant ccRCC cells via the ATF6-dependent ER stress pathway. Xenograft tumors derived from DAPK1-overxpressing ccRCC cells were significantly smaller than the controls in nude mice. Our finding demonstrates that low DAPK1 expression is an independent prognostic indicator that correlates with ccRCC progression and sunitinib resistance.

Background CpG methylation of tumor suppressor genes occurs in the early stage of carcinogenesis. Detecting risk factors for aberrant CpG methylation is clinically important for predicting cancer development. DNA methyltransferase (DNMT) 3a is considered to play critical roles in the DNA methylation process during pathogenesis. In this study, we evaluated the association between DNMT3A polymorphisms (rs6733868 and rs13428812) and CpG methylation status in non-cancerous gastric mucosa. Methods We determined the DNMT3A genotype and CpG methylation status of 4 genes (p14ARF, p16INK4a, DAPK, and CDH1) in 510 subjects without gastric cancer. Helicobacter pylori (HP) infection status was determined by the rapid urease test, urea breath test, speculum examination, or serum antibody test. We determined the DNMT3A genotype using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). CpG methylation status was determined by methylation-specific polymerase chain reaction (MSP). When the methylated band was stronger than 10 ng/μL according to the DNA marker, we judged CpG island hypermethylation (CIHM) to be present. Associations between genotypes and susceptibilities were assessed by logistic regression analysis. Results The minor allele frequencies of both polymorphisms (rs6733868 and rs13428812) were lower in the CpG methylated groups of each of the 4 genes (p14ARF, p16INK4a, DAPK, and CDH1). Using a dominant genetic model, rs6733868 was significantly associated with the hypermethylation of each gene, whereas rs13428812 was associated with the methylation of 3 genes (all except p14ARF). When low-CIHM was defined as 1 or 2 CpG islands methylated and high-CIHM was defined as 3 or more CpG islands methylated, carrying the minor allele of rs6733868 was associated with both decreased low- and high-CIHM, and that of rs13428812 also was associated with a decrease. Comparing low-CIHM with high-CIHM, carrying the minor alleles of rs6733868 or rs13428812 was related to decreased susceptibility to high-CIHM. In HP-infected subjects, carrying the minor alleles of rs6733868 or rs13428812 had a significantly greater association with decreased susceptibility to high-CIHM. Conclusions Our study indicates that polymorphisms of DNMT3A are associated with the accumulation of gene methylation in gastric mucosa. Carrying the minor alleles of rs6733868 or rs13428812 inhibits aberrant gene methylations, which are typically enhanced by HP infection.

OBJECTIVE DNA Methylation of the tumour suppressor gene leading to gene silencing plays an important role in thyroid tumour development. The purpose was to determine the DNA methylation status of phosphatase and tensin homolog (PTEN) and death-associated protein kinase (DAPK) in patients with thyroid nodules and to explore whether they can be used as molecular diagnostic tools to differentiate benign and malignant thyroid nodules. DESIGN, PATIENTS AND MEASUREMENTS Thyroid tissue and blood samples were obtained from normal healthy individuals (controls) and patients suffering from clinically diagnosed thyroid nodular disease [papillary thyroid carcinoma (PTC), adenoma and nodular goitre]. DNA methylation level, mRNA expression and protein expression of PTEN and DAPK in the thyroid tissues and peripheral blood were detected using methylation-specific PCR, semi-quantitative reverse transcription PCR and Western blot, respectively. Diagnostic sensitivity, specific and accuracy of detection were evaluated between blood and thyroid tissue. RESULTS There was a significant increase in the level of DNA methylation of PTEN and DAPK in PTC (P

Significance Tamoxifen is beneficial in treating estrogen receptor–positive breast cancer, but resistance to this treatment eventually ensues. A method to identify mechanisms of tamoxifen resistance identified the histone deacetylase ZIP, leading to the finding that increased expression of the tyrosine kinase JAK2 is one important factor. As a result of this discovery, it may be possible to use an inhibitor of JAK2 to block the aberrant activation of STAT3 caused by ZIP deficiency to help overcome or prevent tamoxifen resistance., Tamoxifen, a widely used modulator of the estrogen receptor (ER), targets ER-positive breast cancer preferentially. We used a powerful validation-based insertion mutagenesis method to find that expression of a dominant-negative, truncated form of the histone deacetylase ZIP led to resistance to tamoxifen. Consistently, increased expression of full-length ZIP gives the opposite phenotype, inhibiting the expression of genes whose products mediate resistance. An important example is JAK2. By binding to two specific sequences in the promoter, ZIP suppresses JAK2 expression. Increased expression and activation of JAK2 when ZIP is inhibited lead to increased STAT3 phosphorylation and increased resistance to tamoxifen, both in cell culture experiments and in a mouse xenograft model. Furthermore, data from human tumors are consistent with the conclusion that decreased expression of ZIP leads to resistance to tamoxifen in ER-positive breast cancer.

Neuroprotective strategies in the treatment of stroke have been attracting a great deal of attentions. Our previous clinical and basic studies have demonstrated that protopanaxadiol ginsenoside-Rd (Rd), a monomer compound extracted from Panax ginseng or Panax notoginseng, has neuroprotective effects against ischemic stroke, probably due to its ability to block Ca2+ overload, an usual consequence of the overactivation of NMDA receptor (NMDAR). As an extending study, we explored here whether Rd exerted its neuroprotection as a novel NMDAR blocker. Our whole-cell patch-clamp results showed that Rd reduced NMDAR currents of cultured rat cortical neurons (EC50 = 7.7 μM) dose-dependently by acting on extrasynaptic NMDAR NR2b subunit. However, unexpectedly, cell transfection and radioligand binding assays revealed that Rd did not bind to the NMDAR channel directly. Alternatively, it inhibited the phosphorylation of NR2b at Ser-1303, a target of death associated protein kinase 1 (DAPK1). Moreover, cell-based and cell-free enzymatic assays showed that Rd did not inhibit the activity of DAPK1 directly, but blocked the activity of calcineurin, a key phosphatase for activating DAPK1. Importantly, other protopanaxadiol ginsenosides were also found to have potential inhibitory effects on calcineurin activity. Furthermore, as expected, calcineurin inhibition by cyclosporin A could mimic Rd’s effects and protect against NMDA-, oxygen glucose deprivation- or transient ischemic stroke-induced neuronal injury. Therefore, our present study provided the first evidence that Rd could exert an inhibitive effect on NMDAR-triggered currents and sequential excitotoxicity through mitigation of DAPK1-mediated NR2b phosphorylation by attenuating calcineurin activity.

Death-associated protein kinase 1 (DAPK1, DAPk, DAPK) is known for its involvement in apoptosis and autophagy-associated cell death. Here, we identified an unexpected function of DAPK1 in suppressing necroptosis. DAPK1-deficiency renders macrophages and dendritic cells susceptible to necroptotic death. We also observed an inhibitory role for DAPK1 in necroptosis in HT-29 cells, since knockdown or knockout of DAPK1 in such cells increased their sensitivity to necroptosis. Increased necroptosis was associated with enhanced formation of the RIPK1–RIPK3–MLKL complex in these DAPK1-deficient cells. We further found that DAPK1-deficiency led to decreased MAPK activated kinase 2 (MK2) activation and reduced RIPK1 S321 phosphorylation, with this latter representing a critical step controlling necrosome formation. Most TNF signaling pathways, including ERK, JNK, and AKT, were not regulated by DAPK. In contrast, DAPK bound p38 MAPK and selectively promoted p38 MAPK activation, resulting in enhanced MK2 phosphorylation. Our results reveal a novel role for DAPK1 in inhibiting necroptosis and illustrate an unexpected selectivity for DAPK1 in promoting p38 MAPK-MK2 activation. Importantly, our study suggests that modulation of necroptosis and p38/MK2-mediated inflammation may be achieved by targeting DAPK1.

Death-associated protein kinase 1 (DAPK1) is a serine/threonine protein kinase that regulates apoptosis and autophagy. DAPK1 is considered to be a therapeutic target for amyloid-β deposition, endometrial adenocarcinomas and acute ischemic stroke. Here, the potent inhibitory activity of the natural anthraquinone purpurin against DAPK1 phosphorylation is shown. Thermodynamic analysis revealed that while the binding affinity of purpurin is similar to that of CPR005231, which is a DAPK1 inhibitor with an imidazopyridazine moiety, the binding of purpurin was more enthalpically favorable. In addition, the inhibition potencies were correlated with the enthalpic changes but not with the binding affinities. Crystallographic analysis of the DAPK1–purpurin complex revealed that the formation of a hydrogen-bond network is likely to contribute to the favorable enthalpic changes and that stabilization of the glycine-rich loop may cause less favorable entropic changes. The present findings indicate that purpurin may be a good lead compound for the discovery of inhibitors of DAPK1, and the observation of enthalpic changes could provide important clues for drug development.

Objective To investigate the impact of death-associated protein kinase 1 (Dapk1) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) via p38MAPK/NF-κB pathway. Methods Dapk1+/+ and Dapk1−/− mice were randomized into Control, LPS, SB203580 (a p38MAPK pathway inhibitor) + LPS, and PDTC (a NF-κB pathway inhibitor) + LPS groups. Cell counts, lung wet to dry weight ratio (W/D weight ratio), as well as indicators of oxidative stress were determined followed by the detection with HE staining, ELISA, qRT-PCR, Western blotting and Immunofluorescence. Besides, to explore whether the effect of Dapk1 on ALI directly mediated via p38MAPK/NF-κB pathway, mice were injected with TC-DAPK 6 (a Dapk1 inhibitor) with or without SB203580/PDTC before LPS administration. Results LPS induced lung injury with increased lung W/D weight ratio, which could be partly reversed by SB203580 and PDTC in LPS-induced mice with activated p38MAPK/NF-κB pathway in lung tissues, especially in Dapk1−/− mice. SB203580 and PDTC reduced total cells and neutrophils in BALF in LPS-induced mice, accompanying with decreased levels of TNF-α, IL-6, MPO, LPO and MDA and the expressions of beclin-1, Atg5 and LC3II, but with the up-regulated activities of SOD and GSH-Px, as well as p62 protein expression. Besides, TC-DAPK 6 aggravated the pathologic injury in LPS-induced ALI with more serious inflammatory response, oxidative stress and autophagy as well as the activated p38MAPK/NF-κB pathway, which were reversed by SB203580 or PDTC. Conclusion Dapk1 improved oxidative stress, inhibited autophagy, and reduce inflammatory response of LPS-induced ALI mice by inhibiting p38MAPK/NF-κB pathway.

Background: Head and neck squamous cell carcinoma (HNSCC) is a significant global burden. The development of a diagnostic or recurrence monitoring test could evolve from the exploitation of molecul...