Inhibition of DAPK3 Suppresses Radiation-Induced Cellular Senescence by Activation of a PGC1α-Dependent Metabolism Pathway in Brain Endothelial Cells.

In the brain, environmental changes, such as neuroinflammation, can induce senescence, characterized by the decreased proliferation of neurons and dendrites and synaptic and vascular damage, resulting in cognitive decline. Senescence promotes neuroinflammatory disorders by senescence-associated secretory phenotypes and reactive oxygen species. In human brain microvascular endothelial cells (HBMVECs), we demonstrate that chronological aging and irradiation increase death-associated protein kinase 3 (DAPK3) expression. To confirm the role of DAPK3 in HBMVEC senescence, we disrupted DAPK3 activity using small interfering RNA (siRNA) or a dominant-negative mutant (DAPK3-P216S), which reduced cellular senescence phenotypes, as assessed by changes in tube formation, senescence-associated beta-galactosidase activity, and cell proliferation. In endothelial cells, DAPK3 promotes cellular senescence by regulating the phosphorylation and inactivation of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) via the protein kinase B pathway, resulting in the decreased expression of mitochondrial metabolism-associated genes, such as ATP5G1, BDNF, and COX5A. Our studies show that DAPK3 is involved in cellular senescence and PGC1α regulation, suggesting that DAPK3 regulation may be important for treating aging-related brain diseases or the response to radiation therapy.

Circ1811 suppresses gastric cancer progression by regulating the miR-632/DAPK1 axis.

Compelling evidence has identified circRNAs as crucial regulators in initiation and progression of various cancers, including gastric cancer (GC). However, the function and regulatory mechanisms of circRNAs in GC remain largely unknown. In this study, attention is paid to a novel circular RNA circ1811, which exerts significant downregulated expression in GC tissues compared with adjacent non-cancerous tissues. The expression of circ1811 in GC tumor tissues is negatively correlated with the extent of lymphatic metastasis in GC patients. Overexpression of circ1811 inhibited GC cell proliferation, migration and invasion while promoting apoptosis, whereas knockdown of circ1811 led to the opposite effects. AGO2 RIP and dual luciferase reporter assays indicated that circ1811 directly sponges miR-632 to upregulate the expression of DAPK1. Collectively, circ1811 acts as a tumor-suppressor for GC progression by regulating the miR-632/DAPK1 axis. Our findings suggest the potential of circ1811 as ideal biomarker and therapeutic target for GC.

The targeting of DAPK1 with miR-190a-3p promotes autophagy in trophoblast cells.

Pre-eclampsia (PE) is a dangerous pathological status that occurs during pregnancy and is a leading reason for both maternal and fetal death. Autophagy is necessary for cellular survival in the face of environmental stress as well as cellular homeostasis and energy management. Aberrant microRNA (miRNA) expression is crucial in the pathophysiology of PE. Although studies have shown that miRNA (miR)-190a-3p function is tissue-specific, the precise involvement of miR-190a-3p in PE has yet to be determined. We discovered that miR-190a-3p was significantly lower and death-associated protein kinase 1 (DAPK1) was significantly higher in PE placental tissues compared to normal tissues, which is consistent with the results in cells. The luciferase analyses demonstrated the target-regulatory relationship between miR-190a-3p and DAPK1. The inhibitory effect of miR-190a-3p on autophagy was reversed by co-transfection of si-DAPK1 and miR-190a-3p inhibitors. Thus, our data indicate that the hypoxia-dependent miR-190a-3p/DAPK1 regulatory pathway is implicated in the development and progression of PE by promoting autophagy in trophoblast cells.

Role of ZIP kinase in development of myofibroblast differentiation from HPMCs.

During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-ß induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-ß significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-ß stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-ß significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-ß. ZIPK gene knockdown (KD) also significantly reduced TGF-ß-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-ß-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs.NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.

CircMAP3K5 promotes cardiomyocyte apoptosis in diabetic cardiomyopathy by regulating miR-22-3p/DAPK2 Axis.

BACKGROUND: Diabetic cardiomyopathy (DCM) is one of the serious complications of the accumulated cardiovascular system in the long course of diabetes. To date, there is no effective treatment available for DCM. Circular RNA (circRNA) is a novel r2egulatory RNA that participates in a variety of cardiac pathological processes. However, the regulatory role of circular RNA MAP3K5 (circMAP3K5) in DCM is largely unclear. METHODS AND RESULTS: Microarray analysis of DCM rats' heart circular RNAs was performed and the highly species-conserved circRNA mitogen-activated protein kinase kinase kinase 5 (circMAP3K5) was identified, which participates in DCM processes. High glucose-provoked cardiotoxicity leads to the up-regulation of circMAP3K5, which mechanistically contributes to cardiomyocyte cell death. Also, in high glucose-induced H9c2 cardiomyocytes, the level of apoptosis was significantly increased, as well as the expression of circMAP3K5. In contrast, the depletion of circMAP3K5 could reduce high glucose-induced apoptosis in cardiomyocytes. In terms of mechanism, circMAP3K5 acts as a miR-22-3p sponge and miR-22-3p directly target death-associated protein kinase 2 (DAPK2) in H9c2 cardiomyocytes, where in circMAP3K5 upregulates DAPK2 expression by targeting miR-22-3p. Moreover, we also found that miR-22-3p inhibitor and pcDNA DAPK2 could antagonize the protective effects brought by the depletion of circMAP3K5. CONCLUSION: CircMAP3K5 is a highly conserved noncoding RNA that is upregulated during DCM process. We concluded that circMAP3K5 promotes high glucose-induced cardiomyocyte apoptosis by regulating the miR-22-3p/DAPK2 axis. The results of this study highlight a novel and translationally important circMAP3K5-based therapeutic approach for DCM.

Quantitative Proteomic and Phosphoproteomic Analyses Reveal a Role of Death-Associated Protein Kinase 1 in Regulating Hippocampal Synapse.

Death-associated protein kinase 1 (DAPK1) is a stress-responsive calcium/calmodulin (CaM)-regulated serine/threonine protein kinase that is actively involved in stress-induced cell death. The dysregulation of DAPK1 has been established in various neurological disorders such as epilepsy, Alzheimer's disease (AD), and Parkinson's disease (PD). Recent research indicates a synaptic localization of DAPK1 in neurons, suggesting a potential role of DAPK1 in modulating synaptic structure and function. However, the key molecules and pathways underlying the influence of DAPK1 on synapses remain elusive. We utilized quantitative proteomic and phosphoproteomic analyses to compare the differences in protein expression and phosphorylation in hippocampal tissues of wild-type (WT) and DAPK1-knockout (KO) mice. Bioinformatic analysis of differentially expressed proteins and phosphoproteins revealed a preferential enrichment of proteins involved in regulating synaptic function, cytoskeletal structure, and neurotransmission. Gene set enrichment analysis (GESA) highlighted altered presynaptic functions including synaptic vesicle priming and glutamate secretion in KO mice. Besides, we observed that proteins with potential phosphorylation motifs of ERK and DAPK1 were overrepresented among the differential phosphoproteins and were highly enriched in neuronal function-related pathways. Furthermore, Western blot analysis validated differences in the expression of several proteins closely associated with presynaptic organization, dendrites and calcium transmembrane transport between KO and WT mice, further corroborating the potential involvement of DAPK1 in the regulation of synaptic functions. Overall, our data provide molecular evidence to elucidate the physiological links between DAPK1 and neuronal functions and help clarify the role of DAPK1 in the pathogenesis of neurodevelopmental and neurodegenerative diseases.

Knockdown of DAPK1 inhibits IL-1ß-induced inflammation and cartilage degradation in human chondrocytes by modulating the PEDF-mediated NF-κB and NLRP3 inflammasome pathway.

Osteoarthritis (OA) is a common joint disease that is characterized by inflammation and cartilage degradation. Death-associated protein kinase 1 (DAPK1) is a multi-domain serine/threonine kinase and has been reported to be involved in the progression of OA. However, its role and mechanism in OA remain unclear. Here, we found the expression of DAPK1 in OA cartilage tissues was higher than that in normal cartilage tissues. The expression of DAPK1 in chondrocytes was up-regulated by IL-1ß. Knockdown of DAPK1 promoted cell viability and anti-apoptotic protein expression, while it inhibited the apoptosis rate and pro-apoptotic protein expressions in IL-1ß-induced chondrocytes. In addition, DAPK1 inhibition reduced the levels of inflammatory cytokines and expressions of matrix metalloproteinases (MMPs), and increased the expressions of collagen II and aggrecan. The data of mechanistic investigation indicated that the expression of pigment epithelium-derived factor (PEDF) was positively regulated by DAPK1. Overexpression of PEDF attenuated the effects of DAPK1 knockdown on IL-1ß-induced cell viability, apoptosis, inflammation, and cartilage degradation. Furthermore, PEDF overexpression restored the activity of the NF-κB pathway and NLRP3 inflammasome after DAPK1 knockdown. Collectively, down-regulation of DAPK1 inhibited IL-1ß-induced inflammation and cartilage degradation via the PEDF-mediated NF-κB and NLRP3 inflammasome pathways.

BNIP3 and DAPK1 methylation in peripheral blood leucocytes are noninvasive biomarkers for gastric cancer.

OBJECTIVE: The objective of this study is to comprehensively investigate the potential value of BNIP3 and DAPK1 methylation in peripheral blood leukocytes as a non-invasive biomarker for the detection of gastric cancer (GC), prediction of chemotherapy efficacy, and prognosis assessment. PATIENTS AND METHODS: Initially, multiple bioinformatic analyses were employed to explore the genetic landscape and biological effects of BNIP3 and DAPK1 in GC tissues. Subsequently, case-control and prospective follow-up studies were conducted to compare the differences in BNIP3 and DAPK1 methylation levels in peripheral blood leukocytes among GC patients and healthy controls, as well as between patients exhibiting sensitivity and resistance to platinum plus fluorouracil treatment, and between patients with varying survival outcomes of GC. Additionally, several predictive nomograms were constructed based on the identified CpG sites and relevant clinical parameters to forecast the occurrence of GC, chemotherapy efficacy, and prognosis. RESULTS: The upregulation of BNIP3 and DAPK1 was found to be associated with the development and poorer survival outcomes of GC. Furthermore, the expression of BNIP3/DAPK1 exhibited an inverse relationship with their DNA methylation levels and demonstrated a positive correlation with immune cell infiltration, as well as the IC50 values of 5-Fluorouracil and Cisplatin in GC tissues. Increased infiltration of macrophages in the high-expression groups was observed to be linked to unfavorable GC survival. In the case-control and follow-up studies, lower methylation levels of BNIP3 and DAPK1 were identified in the peripheral leukocytes of GC patients compared to healthy controls. Hypomethylation was also associated with more aggressive subtypes, diminished chemotherapy efficacy, and poorer survival outcomes in GC. CONCLUSION: The DNA methylation of BNIP3 and DAPK1 in peripheral blood leukocytes holds promise as a novel non-invasive biomarker for predicting the occurrence of GC, chemotherapy efficacy, and prognosis assessment.

Death-associated protein kinase 1 phosphorylates MDM2 and inhibits its protein stability and function.

Breast cancer is one of the major malignancies in women, and most related deaths are due to recurrence, drug resistance, and metastasis. The expression of the mouse double minute 2 (MDM2) oncogene is upregulated in breast cancer; however, its regulatory mechanism has yet to be fully elucidated. Herein, we identified the tumor suppressor death-associated protein kinase 1 (DAPK1) as a novel MDM2 regulator by unbiased peptide library screening. DAPK1 is directly bound to MDM2 and phosphorylates it at Thr419. DAPK1-mediated MDM2 phosphorylation promoted its protein degradation via the ubiquitin-proteasome pathway, resulting in upregulated p53 expression. DAPK1 overexpression, but not its kinase activity-deficient form, decreased colony formation and increased doxorubicin-induced cell death; however, DAPK1 knockdown produced the opposite effects in human breast cancer cells. In a xenograft tumorigenesis assay, DAPK1 overexpression significantly reduced tumor formation, whereas inhibition of DAPK1 kinase activity reduced its antitumorigenic effect. Finally, DAPK1 expression was negatively correlated with MDM2 levels in human breast cancer tissues. Thus, these results suggest that DAPK1-mediated MDM2 phosphorylation and its protein degradation may contribute to its antitumorigenic function in breast cancer.

YTHDF2 promotes gallbladder cancer progression and gemcitabine resistance via m6A-dependent DAPK3 degradation.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic RNA and involved in the carcinogenesis of various malignancies. However, the functions and mechanisms of m6A in gallbladder cancer (GBC) remain unclear. In this study, we investigated the role and underlying mechanism of the RNA-binding protein YT521-B homology domain-containing family protein 2 (YTHDF2), an m6A reader, in GBC. Herein, we detected that YTHDF2 was remarkably upregulated in GBC tissues compared to normal gallbladder tissues. Functionally, YTHDF2 overexpression promoted the proliferation, tumor growth, migration, and invasion of GBC cells while inhibiting the apoptosis in vitro and in vivo. Conversely, YTHDF2 knockdown induced opposite results. Mechanistically, we further investigated the underlying mechanism by integrating RNA immunoprecipitation sequencing (RIP-seq), m6A-modified RIP-seq, and RNA sequencing, which revealed that death-associated protein kinase 3 (DAPK3) is a direct target of YTHDF2. YTHDF2 binds to the 3'-UTR of DAPK3 mRNA and facilitates its degradation in an m6A-dependent manner. DAPK3 inhibition restores the tumor-suppressive phenotype induced by YTHDF2 deficiency. Moreover, the YTHDF2/DAPK3 axis induces the resistance of GBC cells to gemcitabine. In conclusion, we reveal the oncogenic role of YTHDF2 in GBC, demonstrating that YTHDF2 increases the mRNA degradation of the tumor suppressor DAPK3 in an m6A-dependent way, which promotes GBC progression and desensitizes GBC cells to gemcitabine. Our findings provide novel insights into potential therapeutic strategies for GBC.

Mechanism of cAMP Response Element-binding Protein 1/Death-associated Protein Kinase 1 Axis-mediated Hippocampal Neuron Apoptosis in Rat Brain Injury After Cardiopulmonary Resuscitation.

Brain injury represents a leading cause of deaths following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). This study explores the role of CREB1 (cAMP responsive element binding protein 1)/DAPK1 (death associated protein kinase 1) axis in brain injury after CPR. CA was induced by asphyxia in rats, followed by CPR. After CREB1 over-expression, the survival rate and neurological function score of rats were measured. Nissl and TUNEL staining evaluated the pathological condition of hippocampus and apoptosis of hippocampal neurons respectively. H19-7 cells were subjected to OGD/R and infected with oe-CREB1. CCK-8 assay and flow cytometry measured the cell viability and apoptosis. CREB1, DAPK1, and cleaved Caspase-3 expressions were examined using Western blot. The binding between CREB1 and DAPK1 was determined using ChIP and dual-luciferase reporter assays. CREB1 was poorly expressed while DAPK1 was highly expressed in rat hippocampus after CPR. CREB1 overexpression improved rat neurological function, repressed neuron apoptosis, and reduced cleaved Caspase-3 expression. CREB1 was enriched on the DAPK1 promoter and suppressed DAPK1 expression. DAPK1 overexpression reversed the inhibition of OGD/R-insulted apoptosis by CREB1 overexpression. To conclude, CREB1 suppresses hippocampal neuron apoptosis and mitigates brain injury after CPR by inhibiting DAPK1 expression.

The Role of Death-Associated Protein Kinase-1 in Cell Homeostasis-Related Processes.

Tremendous amount of financial resources and manpower have been invested to understand the function of numerous genes that are deregulated during the carcinogenesis process, which can be targeted for anticancer therapeutic interventions. Death-associated protein kinase 1 (DAPK-1) is one of the genes that have shown potential as biomarkers for cancer treatment. It is a member of the kinase family, which also includes Death-associated protein kinase 2 (DAPK-2), Death-associated protein kinase 3 (DAPK-3), Death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK-1) and Death-associated protein kinase-related apoptosis-inducing kinase 2 (DRAK-2). DAPK-1 is a tumour-suppressor gene that is hypermethylated in most human cancers. Additionally, DAPK-1 regulates a number of cellular processes, including apoptosis, autophagy and the cell cycle. The molecular basis by which DAPK-1 induces these cell homeostasis-related processes for cancer prevention is less understood; hence, they need to be investigated. The purpose of this review is to discuss the current understanding of the mechanisms of DAPK-1 in cell homeostasis-related processes, especially apoptosis, autophagy and the cell cycle. It also explores how the expression of DAPK-1 affects carcinogenesis. Since deregulation of DAPK-1 is implicated in the pathogenesis of cancer, altering DAPK-1 expression or activity may be a promising therapeutic strategy against cancer.

Long non-coding RNA nuclear paraspeckle assembly transcript 1 downregulation protects lens epithelial cells from oxidative stress-induced apoptosis by regulating the microRNA-124-3p/death-associated protein kinase 1 axis in age-related cataract.

Oxidative stress plays a significant role in cataract development. It causes the apoptosis of lens epithelial cells (LECs), resulting in lens opacification and accelerating cataract progression. Long non-coding RNAs (lncRNAs) and microRNAs have been linked to cataract development. Notably, lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in LEC apoptosis and cataract formation. However, the molecular mechanism by which NEAT1 causes age-related cataracts remains unknown. In this study, LECs (SRA01/04) were exposed to 200 µM H2O2 to generate an in vitro cataract model. The apoptosis and viability of cells were determined using flow cytometry and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assays, respectively. Additionally, western blotting and quantitative polymerase chain reaction were used to determine the miRNA and lncRNA expression levels. When LECs were treated with hydrogen peroxide, lncRNA NEAT1 expression levels were significantly upregulated, which contributed to LEC apoptosis. Notably, lncRNA NEAT1 suppressed the expression of miR-124-3p, a critical regulator of apoptosis, whereas NEAT1 inhibition increased miR-124-3p expression and alleviated apoptosis. However, this effect was reversed when miR1243p expression was inhibited. Additionally, the miR1243p mimic effectively inhibited the death-associated protein kinase 1 (DAPK1) expression and apoptosis of LECs, while the DAPK1 mimic reversed these effects. In conclusion, our findings indicate that the lncRNA NEAT1/miR-124-3p/DAPK1 signaling loop is involved in the regulation of LEC apoptosis induced by oxidative stress, which can be exploited to develop potential treatment strategies for age-related cataracts.

Death-associated protein kinase 3 regulates the myogenic reactivity of cerebral arteries.

NEW FINDINGS: What is the central question of this study? DAPK3 contributes to the Ca2+ -sensitization of vascular smooth muscle contraction: does this protein kinase participate in the myogenic response of cerebral arteries? What is the main finding and its importance? Small molecule inhibitors of DAPK3 effectively block the myogenic responses of cerebral arteries. HS38-dependent changes to vessel constriction occur independent of LC20 phosphorylation, and therefore DAPK3 appears to operate via the actin cytoskeleton. A role for DAPK3 in the myogenic response was not previously reported, and the results support a potential new therapeutic target in the cerebrovascular system. ABSTRACT: The vascular smooth muscle (VSM) of resistance blood vessels is a target of intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic reactivity of cerebral arteries is critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arteries. DAPK3 is a Ser/Thr kinase involved in Ca2+ -sensitization mechanisms of smooth muscle contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilatation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. A slow return of myogenic tone was observed during the sustained ex vivo exposure of cerebral arteries to HS38. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signalling compensation in response to attenuation of DAPK3 activity. Additional experiments with VSM cells revealed HS38- and siDAPK-dependent effects on the actin cytoskeleton and focal adhesion kinase phosphorylation status. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.

Ablation of Death-Associated Protein Kinase 1 Changes the Transcriptomic Profile and Alters Neural-Related Pathways in the Brain.

Death-associated protein kinase 1 (DAPK1), a Ca2+/calmodulin-dependent serine/threonine kinase, mediates various neuronal functions, including cell death. Abnormal upregulation of DAPK1 is observed in human patients with neurological diseases, such as Alzheimer's disease (AD) and epilepsy. Ablation of DAPK1 expression and suppression of DAPK1 activity attenuates neuropathology and behavior impairments. However, whether DAPK1 regulates gene expression in the brain, and whether its gene profile is implicated in neuronal disorders, remains elusive. To reveal the function and pathogenic role of DAPK1 in neurological diseases in the brain, differential transcriptional profiling was performed in the brains of DAPK1 knockout (DAPK1-KO) mice compared with those of wild-type (WT) mice by RNA sequencing. We showed significantly altered genes in the cerebral cortex, hippocampus, brain stem, and cerebellum of both male and female DAPK1-KO mice compared to those in WT mice, respectively. The genes are implicated in multiple neural-related pathways, including: AD, Parkinson's disease (PD), Huntington's disease (HD), neurodegeneration, glutamatergic synapse, and GABAergic synapse pathways. Moreover, our findings imply that the potassium voltage-gated channel subfamily A member 1 (Kcna1) may be involved in the modulation of DAPK1 in epilepsy. Our study provides insight into the pathological role of DAPK1 in the regulatory networks in the brain and new therapeutic strategies for the treatment of neurological diseases.

Targeting Death-Associated Protein Kinases for Treatment of Human Diseases: Recent Advances and Future Directions.

The death-associated protein kinase (DAPK) family is a member of the calcium/calmodulin-regulated serine/threonine protein kinase family, and studies have shown that its role, as its name suggests, is mainly to regulate cell death. The DAPK family comprises five members, including DAPK1, DAPK2, DAPK3, DRAK1 and DRAK2, which show high homology in the common N-terminal kinase domain but differ in the extra-catalytic domain. Notably, previous research has suggested that the DAPK family plays an essential role in both the development and regulation of human diseases. However, only a few small-molecule inhibitors have been reported. In this Perspective, we mainly discuss the structure, biological function, and role of DAPKs in diseases and the currently discovered small-molecule inhibitors, providing valuable information for the development of the DAPK field.

UR-144, synthetic cannabinoid receptor agonist, induced cardiomyoblast toxicity mechanism comprises cytoplasmic Ca2+ and DAPK1 related autophagy and necrosis.

UR-144, a cannabinoid receptor agonist, is widely used alone or in combination with other synthetic cannabinoids (SCs) all over the world. At overdose, cardiovascular symptoms have been reported and the underlying molecular mechanisms of these adverse effects are not known. It is highly important to clarify the toxic effects of UR-144 for the treatment of poisoning. In the present study, the molecular mechanism of cytotoxic effects of UR-144 is evaluated on a cardiomyoblastic cell line using WST-1 and LDH assays. Apoptosis/necrosis, autophagy, and ROS (reactive oxygen species) levels were determined using flow cytometry. Cytoplasmic Ca2+ levels were measured by using a fluorogenic calcium-binding dye. Released and cytoplasmic troponin T levels, a specific marker of cardiotoxicity, were examined with western blot. For the evaluation of the role of DAPK1, on UR-144-induced cell death, DAPK1 activity and DAPK1 protein level were investigated. Its cytotoxic effects increased in a dose-dependent manner for WST-1 and LDH assays, while membrane damage, one of the signs of necrotic cell death, was more remarkable than damage to mitochondria. Cytoplasmic Ca2+ levels rose after high-dose UR-144 treatment and inhibition of DAPK1 activity ameliorated UR-144-induced cytotoxicity. Released troponin T significantly increased at a dose of 200 µM. ROS and total antioxidant capacity of cells were both reduced following high dose UR-144 treatment. The results indicated that UR-144-induced autophagic and necrotic cell death might be a consequence of elevated cytoplasmic Ca2+ levels and DAPK1 activation. However, in vivo/clinical studies are needed to identify molecular mechanisms of cardiotoxic effects of UR-144.

Death-associated protein kinases and intestinal epithelial homeostasis.

The family of death-associated protein kinases (DAPKs) and DAPK-related apoptosis-inducing protein kinases (DRAKs) act as molecular switches for a multitude of cellular processes, including apoptotic and autophagic cell death events. This review summarizes the mechanisms for kinase activity regulation and discusses recent molecular investigations of DAPK and DRAK family members in the intestinal epithelium. In general, recent literature convincingly supports the importance of this family of protein kinases in the homeostatic processes that govern the proper function of the intestinal epithelium. Each of the DAPK family of proteins possesses distinct biochemical properties, and we compare similarities in the information available as well as those cases where functional distinctions are apparent. As the prototypical member of the family, DAPK1 is noteworthy for its tumor suppressor function and association with colorectal cancer. In the intestinal epithelium, DAPK2 is associated with programmed cell death, potential tumor-suppressive functions, and a unique influence on granulocyte biology. The impact of the DRAKs in the epithelium is understudied, but recent studies support a role for DRAK1 in inflammation-mediated tumor growth and metastasis. A commentary is provided on the potential importance of DAPK3 in facilitating epithelial restitution and wound healing during the resolution of colitis. An update on efforts to develop selective pharmacologic effectors of individual DAPK members is also supplied.

MiR-191-5p Attenuates Tau Phosphorylation, Aß Generation, and Neuronal Cell Death by Regulating Death-Associated Protein Kinase 1.

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 (Aß) 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, Aß secretion, and neuronal cell death by directly targeting DAPK1, providing an attractive therapeutic option for AD.

Knockdown of DAPK1 attenuates IL-1ß-induced extracellular matrix degradation and inflammatory response in osteoarthritis chondrocytes via regulating the p38 MAPK-signaling pathway.

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)-1ß-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-1ß were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution, 5-ethynyl-2ß-deoxyuridine staining and flow cytometry. The chondrocyte degradation and inflammatory response in IL-1ß-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-1ß-induced chondrocytes. Down-regulation of DAPK1 enhanced IL-1ß-induced chondrocyte proliferation. DAPK1 knockdown inhibited IL-1ß-induced chondrocyte degradation. In addition, DAPK1 depletion inhibited IL-1ß-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-1ß-induced extracellular matrix degradation and inflammatory response in OA chondrocytes by regulating the p38 MAPK pathway.