Death-associated Protein Kinase mRNA Research Articles

Mechanism study of 6-shogaol alleviating cerebral ischemia/reperfusion injury by regulating microRNA-26a-5p/death-associated protein kinase 1

To investigate whether 6-shogaol (6-SH) alleviates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal autophagy and calcium overload by promoting the expression of microRNA-26a-5p (miR-26a-5p) and inhibiting death-associated protein kinase 1 (DAPK1), and to explore its potential mechanisms. Primary cultured logarithmic growth phase mouse hippocampal neurons HT22 cells were taken and cell counting kit-8 (CCK-8) was used to detect cell viability, searching for the optimal concentration of Na2S2O4. HT22 cells were divided into blank control group (NC group), OGD/R group (sugar-free culture medium + 10 mmol/L Na2S2O4 treatment for 1.5 hours followed by normal culture medium for 4 hours), 6-SH intervention group (cultured with 10 μmol/L 6-SH for 4 hours after OGD), negative control inhibitor pretreatment group (transfected with negative control inhibitor for 48 hours followed by OGD, then cultured with 6-SH for 4 hours), and miR-26a-5p inhibitor pretreatment group (transfected with miR-26a-5p inhibitor for 48 hours followed by OGD, then cultured with 6-SH for 4 hours). Cell viability of each group was detected by CCK-8 method; cell ultrastructure was observed under transmission electron microscopy; real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expressions of DAPK1 and miR-26a-5p; molecular docking were used to verify the interaction between 6-SH and miR-26a-5p; dual-luciferase assay was used to verify the targeting relationship between DAPK1 and miR-26a-5p; flow cytometry was used to determine the levels of intracellular Ca2+; Western blotting was used to detect the protein expressions of phosphorylated-glutamate receptor 2B (p-NMDAR2B) Ser1303, DAPK1, autophagy related protein Beclin1, light chain 3 (LC3), and p-DAPK1 Ser308; immunofluorescence was used to detect the expression of LC3 and Beclin1. The results of the CCK-8 assay showed that the cell viability of the 6-SH intervention group was significantly increased compared to the OGD/R group, while the cell viability of the miR-26a-5p inhibitor pretreatment group was significantly decreased compared to the 6-SH intervention group. Transmission electron microscopy revealed that the number of autophagosomes in the 6-SH intervention group was significantly reduced compared to the OGD/R group, while the number of autophagosomes in the miR-26a-5p inhibitor pretreatment group was significantly increased compared to the 6-SH intervention group. RT-qPCR results showed that compared with the OGD/R group, the expression of miR-26a-5p was significantly upregulated and the expression of DAPK1 mRNA was significantly downregulated in the 6-SH intervention group; compared with the 6-SH intervention group, the expression of miR-26a-5p was significantly downregulated and the expression of DAPK1 mRNA was significantly upregulated in the miR-26a-5p inhibitor pretreatment group. Molecular docking verified the interaction between 6-SH and miR-26a-5p. Dual-luciferase reporter gene assay showed that compared with the negative control group, mmu-miR-26a-5p significantly downregulated the luciferase expression of m-DAPK1-3UTR-WT, indicating a binding interaction between them. Flow cytometry results showed that compared with the OGD/R group, the level of intracellular Ca2+; was significantly decreased in the 6-SH intervention group; compared with the 6-SH intervention group, the level of Ca2+ was significantly increased in the miR-26a-5p inhibitor pretreatment group. Western blotting results showed that compared with the OGD/R group, the protein expressions of p-NMDAR2B Ser1303, DAPK1, Beclin1, and LC3 were significantly decreased in the 6-SH intervention group (p-NMDAR2B Ser1303/β-actin: 2.34±0.27 vs. 4.78±0.39, DAPK1/β-actin: 1.40±0.13 vs. 2.37±0.21, Beclin1/β-actin: 2.61±0.32 vs. 4.32±0.29, LC3/β-actin: 2.52±0.45 vs. 5.09±0.18, all P < 0.05), while the protein expression of p-DAPK1 Ser308 was significantly increased (p-DAPK1 Ser308/β-actin: 0.66±0.09 vs. 0.40±0.02, P < 0.05); compared with the 6-SH intervention group, the protein expressions of p-NMDAR2B Ser1303, DAPK1, Beclin1, and LC3 were significantly increased in the miR-26a-5p inhibitor pretreatment group (p-NMDAR2B Ser1303/β-actin: 4.08±0.14 vs. 2.34±0.27, DAPK1/β-actin: 1.96±0.15 vs. 1.40±0.13, Beclin1/β-actin: 3.92±0.31 vs. 2.61±0.32, LC3/β-actin: 4.33±0.33 vs. 2.52±0.45, all P < 0.05), while the expression of p-DAPK1 Ser308 protein was significantly decreased (p-DAPK1 Ser308/β-actin: 0.33±0.12 vs. 0.66±0.09, P < 0.05); immunofluorescence staining showed that compared with the OGD/R group, the fluorescence intensity of LC3 and Beclin1 was significantly decreased in the 6-SH intervention group; compared with the 6-SH intervention group, the fluorescence intensity of LC3 and Beclin1 was significantly increased in the miR-26a-5p inhibitor pretreatment group. 6-SH can alleviate neuronal damage by regulating miR-26a-5p/DAPK1 to reduce autophagy and calcium overload in cells.

MiR-143-3p Inhibits Aberrant Tau Phosphorylation and Amyloidogenic Processing of APP by Directly Targeting DAPK1 in Alzheimer’s Disease

The neuropathology of Alzheimer’s disease (AD) is characterized by intracellular aggregation of hyperphosphorylated tau and extracellular accumulation of beta-amyloid (Aβ). Death-associated protein kinase 1 (DAPK1), as a novel therapeutic target, shows promise for the treatment of human AD, but the regulatory mechanisms of DAPK1 expression in AD remain unclear. In this study, we identified miR-143-3p as a promising candidate for targeting DAPK1. miR-143-3p directly bound to the 3′ untranslated region of human DAPK1 mRNA and inhibited its translation. miR-143-3p decreased tau phosphorylation and promoted neurite outgrowth and microtubule assembly. Moreover, miR-143-3p attenuated amyloid precursor protein (APP) phosphorylation and reduced the generation of Aβ40 and Aβ42. Furthermore, restoring DAPK1 expression with miR-143-3p antagonized the effects of miR-143-3p in attenuating tau hyperphosphorylation and Aβ production. In addition, the miR-143-3p levels were downregulated and correlated inversely with the expression of DAPK1 in the hippocampus of AD patients. Our results suggest that miR-143-3p might play critical roles in regulating both aberrant tau phosphorylation and amyloidogenic processing of APP by targeting DAPK1 and thus offer a potential novel therapeutic strategy for AD.

Bone mesenchymal stem cell-derived extracellular vesicles inhibit DAPK1-mediated inflammation by delivering miR-191 to macrophages

Alveolar macrophage activation and apoptosis are vital contributors to sepsis-associated acute lung injury (ALI). However, the mechanisms of alveolar macrophage activation are yet to be clarified. Death-associated protein kinase 1 (DAPK1) is one of the potential candidates that play crucial roles in regulating alveolar macrophage inflammation. Herein, we found that primary human bone mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) antagonize LPS-induced inflammation in the THP-1 human macrophage-like cell line. Mechanistically, LPS stimulation elevates the expression of DAPK1 and the inflammation markers in THP-1 cells, while BMSC-derived EVs inhibit the expression of DAPK1 and inflammation through delivering miR-191, which can target the 3′-UTR of the DAPK1 mRNA and therefore suppress its translation. The importance of DAPK1 in the activation of THP-1 is also stressed in this study. Our findings provide evidence that BMSC-derived EVs regulate the alveolar macrophage inflammation and highlight BMSC-derived EVs as a potential vehicle to deliver biomacromolecules to macrophages.

Low DAPK1 expression correlates with poor prognosis and sunitinib resistance in clear cell renal cell carcinoma.

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.

Death associated protein kinase 1 (DAPK-1) is increased in preeclampsia

IntroductionDeath associated protein kinase-1 (DAPK-1) is highly expressed in the placenta relative to all other human tissues. We examine whether it is differentially expressed with preeclampsia. MethodsWe examined samples from a large prospective collection of plasma from 2002 women. We split the samples into two cohorts: Cohort 1 (n = 1000) and Cohort 2 (n = 1002). We first measured circulating DAPK-1 at 36 weeks' gestation in a nested case-control group (from Cohort 1) of 39 women who developed preeclampsia and 98 controls. We then validated our findings by measuring circulating levels in all samples from both cohorts. We also measured DAPK-1 in the circulation and placentas of women who were diagnosed with preterm preeclampsia or delivered a growth restricted infant at <34 weeks’ gestation. ResultsIn the case-control study, circulating DAPK-1 was significantly increased in women destined to develop preeclampsia (p < 0.01). We validated this by measuring circulating levels in Cohorts 1 and 2. Again, circulating DAPK-1 was significantly higher (p < 0.001) among women destined to develop preeclampsia (Cohort 1, Area under the receiver operator characteristic curve (AUC) = 0.66; Cohort 2 AUC = 0.67). Circulating DAPK-1 was also significantly elevated in women with established preterm preeclampsia. Placental DAPK-1 mRNA and protein expression were elevated in women with established preeclampsia. DiscussionDAPK-1 is a novel placenta-enriched molecule that is elevated in the circulation of women preceding the diagnosis of preeclampsia and is likely to be secreted from the placenta.

Expression and Functional Relevance of Death-Associated Protein Kinase in Human Drug-Resistant Epileptic Brain: Focusing on the Neurovascular Interface

Death-associated protein kinase (DAPK) is a key player in various cell death signaling pathways. Prolonged seizures induce neuronal stress; thus, we studied DAPK expression in resected brain tissues from patients with refractory epilepsy and the pathophysiological relevance of neurovascular DAPK. We used brain resections from temporal lobe epilepsy (TLE), tumor (BT), arteriovenous malformation (AVM), and autopsy, and isolated human endothelial cells (EPI-ECs) and glial cells (EPI-Astro) from epileptic brains compared to control brain endothelial cells (HBMECs) and astrocytes. DAPK and phosphorylated DAPK (p-DAPK) expression was evaluated by immunohistochemistry and western blot. Subcellular localization of DAPK in epileptic brain was explored; DAPK mRNA/protein levels in EPI-ECs/EPI-Astro were evaluated. We assessed DAPK localization with hypoxic inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) in epilepsy, BT, and AVM. We found DAPK overexpression across neurons, microcapillaries, and astrocytes in TLE vs controls; DAPK and p-DAPK levels significantly increased only in microsomal fractions of epileptic brain. DAPK mRNA remained unchanged, although increased DAPK and p-DAPK protein expression was observed in EPI-ECs. DAPK inhibition reduced p-DAPK, HIF-1α, and VEGF expression, but increased cytotoxicity and decreased cell viability in EPI-ECs and EPI-astro vs. controls. DAPK staining in TLE resembled BT and AVM, with predominant DAPK/p-DAPK expression in neurons and vasculature. Taken together, these findings suggest DAPK could be a potential molecular target in neuronal death and vascular changes in epilepsy. Increased brain endothelial and astrocytic DAPK in epilepsy, identified for the first time, may have relevance to angiogenesis, hypoxia, and cell survival in pathological conditions.

The “all-or-none” period: is it 2 weeks?

Death associated protein kinase-1 (DAPK-1) is highly expressed in the placenta relative to all other human tissues. We examine whether it is differentially expressed with preeclampsia.We examined samples from a large prospective collection of plasma from 2002 women. We split the samples into two cohorts: Cohort 1 (n = 1000) and Cohort 2 (n = 1002). We first measured circulating DAPK-1 at 36 weeks' gestation in a nested case-control group (from Cohort 1) of 39 women who developed preeclampsia and 98 controls. We then validated our findings by measuring circulating levels in all samples from both cohorts. We also measured DAPK-1 in the circulation and placentas of women who were diagnosed with preterm preeclampsia or delivered a growth restricted infant at <34 weeks’ gestation.In the case-control study, circulating DAPK-1 was significantly increased in women destined to develop preeclampsia (p < 0.01). We validated this by measuring circulating levels in Cohorts 1 and 2. Again, circulating DAPK-1 was significantly higher (p < 0.001) among women destined to develop preeclampsia (Cohort 1, Area under the receiver operator characteristic curve (AUC) = 0.66; Cohort 2 AUC = 0.67). Circulating DAPK-1 was also significantly elevated in women with established preterm preeclampsia. Placental DAPK-1 mRNA and protein expression were elevated in women with established preeclampsia.DAPK-1 is a novel placenta-enriched molecule that is elevated in the circulation of women preceding the diagnosis of preeclampsia and is likely to be secreted from the placenta.

Identification of DAPK1 Promoter Hypermethylation as a Biomarker for Intra-Epithelial Lesion and Cervical Cancer: A Meta-Analysis of Published Studies, TCGA, and GEO Datasets.

Background: Promoter hypermethylation in death-associated protein kinase 1 (DAPK1) gene has been long linked to cervical neoplasia, but the established results remained controversial. Here, we performed a meta-analysis to assess the associations of DAPK1 promoter hypermethylation with low-grade intra-epithelial lesion (HSIL), high-grade intra-epithelial lesion (HSIL), cervical cancer (CC), and clinicopathological features of CC.Methods: Published studies with qualitative methylation data were initially searched from PubMed, Web of Science, EMBASE, and China National Knowledge Infrastructure databases (up to March 2018). Then, quantitative methylation datasets, retrieved from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, were pooled to validate the results of published studies.Results: In a meta-analysis of 37 published studies, DAPK1 promoter hypermethylation progressively increased the risk of LSIL by 2.41-fold (P = 0.012), HSIL by 7.62-fold (P < 0.001), and CC by 23.17-fold (P < 0.001). Summary receiver operating characteristic curves suggested a potential diagnostic value of DAPK1 promoter hypermethylation in CC, with a large area-under-the-curve of 0.83, a high specificity of 97%, and a moderate sensitivity of 59%. There were significant impacts of DAPK1 promoter hypermethylation on histological type (odds ratio (OR) = 3.53, P < 0.001) and FIGO stage of CC (OR = 2.15, P = 0.003). Then, a pooled analysis of nine TCGA and GEO datasets, covering 13 CPG sites within DAPK1 promoter, identified eight CC-associated sites, six sites with diagnostic values for CC (pooled specificities: 74–90%; pooled sensitivities: 70–81%), nine loci associated with the histological type of CC, and all 13 loci with down-regulated effects on DAPK1 mRNA expression.Conclusion: The meta-analysis suggests that DAPK1 promoter hypermethylation is significantly associated with the disease severity of cervical neoplasia. DAPK1 methylation detection exhibits a promising ability to discriminate CC from cancer-free controls.

DAPK1 as an independent prognostic marker in liver cancer.

The death-associated protein kinase 1 (DAPK1) can act as an oncogene or a tumor suppressor gene depending on the cellular context as well as external stimuli. Our study aims to investigate the prognostic significance of DAPK1 in liver cancer in both mRNA and protein levels. The mRNA expression of DAPK1 was extracted from the Gene Expression Omnibus database in three independent liver cancer datasets while protein expression of DAPK1 was detected by immunohistochemistry in our Chinese liver cancer patient cohort. The associations between DAPK1 expression and clinical characteristics were tested. DAPK1 mRNA expression was down-regulated in liver cancer. Low levels of DAPK1 mRNA were associated with shorter survival in a liver cancer patient cohort (n = 115; p = 0.041), while negative staining of DAPK1 protein was significantly correlated with shorter time to progression (p = 0.002) and overall survival (p = 0.02). DAPK1 was an independent prognostic marker for both time to progression and overall survival by multivariate analysis. Liver cancer with the b-catenin mutation has a lower DAPK1 expression, suggesting that DAPK1 may be regulated under the b-catenin pathway. In addition, we also identified genes that are co-regulated with DAPK1. DAPK1 expression was positively correlated with IRF2, IL7R, PCOLCE and ZBTB16, and negatively correlated with SLC16A3 in both liver cancer datasets. Among these genes, PCOLCE and ZBTB16 were significantly down-regulated, while SLC16A3 was significantly upregulated in liver cancer. By using connectivity mapping of these co-regulated genes, we have identified amcinonide and sulpiride as potential small molecules that could potentially reverse DAPK1/PCOLCE/ZBTB16/SLC16A3 expression. Our study demonstrated for the first time that both DAPK1 mRNA and protein expression levels are important prognostic markers in liver cancer, and have identified genes that may contribute to DAPK1-mediated liver carcinogenesis.

MicroRNA-141-3p targets DAPK1 and inhibits apoptosis in rat ovarian granulosa cells.

The polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disorder. MicroRNAs negatively regulate the expression of target genes at posttranscriptional level by binding to the 3' untranslated region of target genes. Our previous study showed that miR-141-3p was dramatically decreased in the ovaries of rat PCOS models. In this study, we aimed to characterize the target of miR-141-3p in rat ovarian granulosa cells. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay showed that cell viability was dramatically increased when miR-141-3p was overexpressed but was decreased when miR-141-3p was interfered. Flow cytometry showed that cell apoptotic rate was dramatically decreased when miR-141-3p was overexpressed but was increased when miR-141-3p was interfered. Bioinformatics analysis predicted that death-associated protein kinase 1 (DAPK1) might be the target gene of miR-141-3p because the 3' untranslated region of DAPK1 contains sequences complementary to microRNA-141-3p. Transfection with miR-141-3p mimics and inhibitor into granulosa cells showed that both DAPK1 mRNA and protein levels were negatively correlated with miR-141-3p level. Dual-luciferase reporter assay established that DAPK1 was the target of miR-141-3p. Taken together, our data indicate that miR-141-3p may inhibit ovarian granulosa cell apoptosis via targeting DAPK1 and is involved in the etiology of PCOS.

Promoter methylation and expression of death-associated protein kinase gene in acute leukemia

To study the clinical implications of death-associated protein kinase (DAPK) promoter methylation and DAPK gene expression in untreated patients with acute leukemia. Methylation-specific PCR and RT-PCR were employed to detect the DAPK gene methylation and mRNA expression in the bone marrow of 60 patients with acute myeloid leukemia (AML), 55 patients with acute lymphocytic leukemia (ALL), and 17 normal subjects. The positivity rate of DAPK methylation was significantly higher in ALL patients (29.1%) than in AML patients group (5%) and normal subjects (0%) (P<0.01). No correlation was found between DAPK gene methylation and the clinical features in ALL patients (P>0.05). DAPK mRNA expression level differed significantly among the 3 groups (P=0.000), and was the highest in normal subjects and the lowest in ALL patients. In ALL patients, the expression level of DAPK mRNA showed a significant inverse correlation with DAPK promoter methylation (P<0.05). The methylation rate of DAPK gene is higher in untreated ALL patients than in AML patients and normal subjects. DAPK gene methylation is not correlated with the clinical features of ALL patients but is probably related with the low gene expression level of DAPK in these patients.

Placental DAPK1 and autophagy marker LC3B-II are dysregulated by TNF-\u03b1 in a gestational age-dependent manner

Autophagy, a cell-survival process responsible for degradation of protein aggregates and damaged organelles, is increasingly recognized as another mechanism essential for human placentation. A substantial body of experiments suggests inflammation and oxidative stress as the underlying stimuli for altered placental autophagy, giving rise to placenta dysfunction and pregnancy pathologies. Here, the hypothesis is tested whether or not pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α are able to influence the expression profile of autophagy genes in human first-trimester villous placenta. Autophagy-focused qPCR arrays identified substantial downregulation of death-associated protein kinase 1 (DAPK1) in first-trimester placental explants in response to IL-6 and TNF-α, respectively. Immunohistochemistry of placental explants detected considerable DAPK1 staining in placental macrophages, villous cytotrophoblasts and less intense in the syncytiotrophoblast. Both immunohistochemistry and Western blot showed decreased DAPK1 protein in TNF-α-treated placental explants compared to control. On cellular level, DAPK1 expression decreased in SGHPL-4 trophoblasts in response to TNF-α. Observed changes in the expression profile of autophagy-related genes were reflected by significantly decreased lipidation of autophagy marker microtubule-associated protein light chain 3 beta (LC3B-II) in first trimester placental explants in response to TNF-α. Analysis of TNF-α-treated term placental explants showed decreased DAPK1 protein, whereas in contrast to first-trimester LC3B expression and lipidation increased. Immunohistochemistry of placental tissues from early-onset preeclampsia (PE) showed less DAPK1 staining, when compared to controls. Accordingly, DAPK1 mRNA and protein were decreased in primary trophoblasts isolated from early-onset PE, while LC3B-I and -II were increased. Results from this study suggest that DAPK1, a regulator of apoptosis, autophagy and programmed necrosis, decreases in human placenta in response to elevated maternal TNF-α, irrespective of gestational age. In contrast, TNF-α differentially regulates levels of autophagy marker LC3B in human placenta over gestation.

Overexpression of microRNA-133a inhibits ischemia-reperfusion-induced cardiomyocyte apoptosis by targeting DAPK2.

To examine microRNA-133a (miR-133a) endogenous expression in cardiomyocytes after ischemia-reperfusion (I/R) injury and study the effects of miR-133a overexpression on I/R injury-induced cardiomyocyte apoptosis. Dual-Luciferase Reporter Assay detected dynamic expression of miR-133a. In an in vitro hypoxia-reoxygenation (HR) injury model and an in vivo rat model of I/R injury, rat cardiomyocytes were transfected with miR-133a mimic to test the effects of miR-133a overexpression on apoptosis. MiR-133a and Death Associated Protein Kinase 2 (DAPK2) mRNA expression was measured using real-time-PCR, and DAPK2 protein expression was detected by western blotting. Annexin V-fluorescein isothiocyanate/propidium iodide (PI) double-staining measured the apoptosis rate in H9C2 cells and transferase dUTP nick end labeling assay quantified the cardiomyocyte apoptosis rate in tissues obtained from in vivo the rat model. DAPK2 is a target of miR-133a. Both in vitro and in vivo results confirmed that after expression of miR-133a mimics, miR-133a levels increased, which was accompanied by decrease in DAPK2 mRNA and protein expression. In H9C2 cells, HR injury caused a sharp decrease in miR-133a expression and a significant upregualtion of DAPK2 mRNA and protein levels. However, exogenous miR-133a expression led to a significant reduction in DAPK2 mRNA and protein levels despite HR injury. Similar results were obtained from in vivo I/R injury model. After HR injury or I/R injury the apoptosis rate of myocardial cells was highly elevated and decreased significantly only after transfection of miR-133a into cardiomyocytes. MiR-133a overexpression may inhibit I/R injury-mediated cardiomyocyte apoptosis by targeting DAPK2, leading to reduced DAPK2 protein, thus miR-133a may potentially have a high therapeutic value in I/R injury.

DAPK2 Downregulation Associates With Attenuated Adipocyte Autophagic Clearance in Human Obesity.

Adipose tissue dysfunction in obesity has been linked to low-grade inflammation causing insulin resistance. Transcriptomic studies have identified death-associated protein kinase 2 (DAPK2) among the most strongly downregulated adipose tissue genes in human obesity, but the role of this kinase is unknown. We show that mature adipocytes rather than the stromal vascular cells in adipose tissue mainly expressed DAPK2 and that DAPK2 mRNA in obese patients gradually recovered after bariatric surgery-induced weight loss. DAPK2 mRNA is also downregulated in high-fat diet-induced obese mice. Adenoviral-mediated DAPK2 overexpression in 3T3-L1 adipocytes did not affect lipid droplet size or cell viability but did increase autophagic clearance in nutrient-rich conditions, dependent on protein kinase activity. Conversely, DAPK2 inhibition in human preadipocytes by small interfering RNA decreased LC3-II accumulation rates with lysosome inhibitors. This led us to assess autophagic clearance in adipocytes freshly isolated from subcutaneous adipose tissue of obese patients. Severe reduction in autophagic flux was observed in obese adipocytes compared with control adipocytes, inversely correlated to fat cell lipids. After bariatric surgery, adipocyte autophagic clearance partially recovered proportional to the extent of fat cell size reduction. This study links adipocyte expression of an autophagy-regulating kinase, lysosome-mediated clearance and fat cell lipid accumulation; it demonstrates obesity-related attenuated autophagy in adipocytes, and identifies DAPK2 dependence in this regulation.

Lysine Malonylation as a New Inflammatory Signal

Malonylation is a recently uncovered protein post-translational modification. It involves the attachment of a malonyl group to lysines, resulting in an extra 86 Da mass, as well as a change in charge comparable to that of phosphorylation. The enzyme responsible for malonylation has not yet been uncovered, but it seems like malonylation levels are dependent on malonyl-CoA concentration. The discovery of this new modification took place during a time in which immunometabolism became an emerging field and many studies into the metabolism of immune cells have recently highlighted the tight link existing between the metabolic status and the function of these cells. This led us to investigate whether protein malonylation might be a new mechanism by which the cell's metabolism can control the inflammatory response. We have been able to show that protein malonylation can be induced in macrophages following activation with various toll-like receptor ligands. In addition, the TLR4 ligand, LPS, can specifically increase malonylation of the glycolytic enzyme GAPDH in macrophages. Through mass-spec analysis we identified a single malonylated lysine present in GAPDH in resting macrophages, while two were identified following activation with LPS. Interestingly, GAPDH enzymatic activity increases under the same conditions. Furthermore, as part of the GAIT complex, GAPDH is capable of binding to the 3'-UTR region of the death-associated protein kinase (DAPK) mRNA and inhibit its translation. Our data would suggest that following activation, GAPDH releases DAPK mRNA. Overall, our results suggest that protein malonylation is an important signal for LPS signaling in macrophages, that may be responsible for controlling the functions of the glycolytic enzyme GAPDH following macrophage activation. This research is supported by Science Foundation Ireland.

Hypermethylation of the death-associated protein kinase CpG island in canine B-cell lymphoid tumors

Death-associated protein kinase (DAPK) is a 160-kD serine/threonine kinase known as a key molecule in interferon-γ (IFN-γ)-induced apoptosis and tumor suppression. Hypermethylation of the CpG island in DAPK inactivates the gene in a variety of human malignancies. This study aimed to detect the inactivation of DAPK in canine lymphoid tumor cells. The sequence of canine DAPK cDNA was obtained from normal dog peripheral blood mononuclear cells after reverse transcription polymerase chain reaction (RT-PCR). By rapid amplification of 5′-cDNA ends, the transcription initiation site of the DAPK gene was identified. The CpG island located upstream of the translation initiation site was identified by using a search algorithm. The methylation status of the CpG island was examined using bisulfite sequence analysis and methylation-specific PCR (MSP). The inactivation of DAPK gene was examined in 3 canine lymphoid tumor cell lines, GL-1 (B-cell leukemia), CLBL-1 (B-cell lymphoma), and CL-1 (T-cell lymphoma). DAPK mRNA expression was measured by real-time RT-PCR. IFN-γ-induced apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. The influence of demethylation was examined with 5-aza-2′-deoxycytidine (5-aza-dC). The methylation status in 14 dogs with various lymphoid tumors was screened by MSP. A 1926-bp CpG island containing 280 CpG repeats was identified upstream of the translation start site of canine DAPK. Bisulfate sequence analysis and MSP revealed hypermethylation of the CpG island in GL-1 cells, but not in CLBL-1 or CL-1 cells. The amount of DAPK mRNA was significantly smaller in GL-1 cells than CLBL-1 and CL-1 cells. IFN-γ-induced apoptosis was detected in CLBL-1 and CL-1 cells but not in GL-1 cells. Treatment with 5-aza-dC significantly increased the amount of DAPK mRNA and IFN-γ-induced apoptosis in GL-1 cells. These results revealed the inactivation of DAPK through methylation of its CpG island in GL-1 cells. MSP showed hypermethylation of the DAPK CpG island in 5 of 8 primary B-cell lymphoma samples, but not in any of the 6 primary T-cell lymphoid tumor samples obtained from canine patients. DAPK was inactivated through hypermethylation of its CpG island in canine B-cell lymphoid tumor cells. This study will lead to the use of canine B-cell lymphoid tumors as an animal model to evaluate the efficacy of demethylating agents.

Expression of death-associated protein kinase gene and methylation status of promoter region in acute leukemia

This study was purpose to investigate the expression of death-associated protein kinase (DAPK) gene in acute leukemia (AL) patients and the methylation status of its promoter region through experiments of DAPK methylation and expression, and to analyze the relation between them. The expression of DAPK gene in leukemia cells and normal bone marrow cells was detected by RT-PCR; the methylation status of DAPK gene promoter region in cells from AL patients and leukemia cell lines HL-60 and U937 was detected by nested methylation specific PCR (n-MSP); 2 randomly primers selected from randomly amplified products of second round nMS-PCR were cloned and sequenced in professional company. The results showed that the DAPK gene expressed in bone marrow specimens of all 10 normal controls, with average value of expression 0.92 ± 0.18, while the average value of DAPK expression in bone marrow specimens of AL patients was 0.61 ± 0.40 which was lower than that in normal controls (P < 0.05). The low or deletion of DAPK mRNA expression were found in bone marrow specimens of 9/17 (52.94%) cases of ALL and 42/102 (41.18%) cases of AML. The cell line U937 showed normal expression of DAPK gene, while cell line HL-60 showed the expression detection of DAPK gene. The methylation of DAPK promoter region existed in 33 out of bone marrow specimens of 102 AML patients and in 8 out of bone marrow specimens of 17 ALL patients, the methylation rates were 32.4% (33/102) and 47% respectively. The DAPK promoter region in bone marrow of 7 normal controls was unmethylated, while DAPK promoter region in U937 cells and HL-60 cells were unmethylated and methylated respectively. The DAPK mRNA expression in ALL and AML patients significantly negatively correlated with the methylation of its promoter region (r = -0.855, P < 0.05, in AML patients and r = -0.343, P < 0.05, in AML patients) suggesting the close relationship between them. It is concluded that the methylation of DAPK gene promoter region relates with abnormal expression or detection of DAPK mRNA in AL patients.

DAPK-HSF1 interaction as a positive-feedback mechanism stimulating TNF-induced apoptosis in colorectal cancer cells.

Death-associated protein kinase (DAPK) is a serine-threonine kinase with tumor suppressor function. Previously, we demonstrated that tumor necrosis factor (TNF) induced DAPK-mediated apoptosis in colorectal cancer. However, the protein-protein interaction network associated with TNF-DAPK signaling still remains unclear. We identified HSF1 as a new DAPK phosphorylation target in response to low concentrations of TNF and verified a physical interaction between DAPK and HSF1 both in vitro and in vivo. We show that HSF1 binds to the DAPK promoter. Transient overexpression of HSF1 protein led to an increase in DAPK mRNA level and consequently to an increase in the amount of apoptosis. By contrast, treatment with a DAPK-specific inhibitor as well as DAPK knockdown abolished the phosphorylation of HSF1 at Ser230 (pHSF1(Ser230)). Furthermore, translational studies demonstrated a positive correlation between DAPK and pHSF1(Ser230) protein expression in human colorectal carcinoma tissues. Taken together, our data define a novel link between DAPK and HSF1 and highlight a positive-feedback loop in DAPK regulation under mild inflammatory stress conditions in colorectal tumors. For the first time, we show that under TNF the pro-survival HSF1 protein can be redirected to a pro-apoptotic program.

Death Associated Protein Kinases: Molecular Structure and Brain Injury

Perinatal brain damage underlies an important share of motor and neurodevelopmental disabilities, such as cerebral palsy, cognitive impairment, visual dysfunction and epilepsy. Clinical, epidemiological, and experimental studies have revealed that factors such as inflammation, excitotoxicity and oxidative stress contribute considerably to both white and grey matter injury in the immature brain. A member of the death associated protein kinase (DAPk) family, DAPk1, has been implicated in cerebral ischemic damage, whereby DAPk1 potentiates NMDA receptor-mediated excitotoxicity through interaction with the NR2BR subunit. DAPk1 also mediate a range of activities from autophagy, membrane blebbing and DNA fragmentation ultimately leading to cell death. DAPk mRNA levels are particularly highly expressed in the developing brain and thus, we hypothesize that DAPk1 may play a role in perinatal brain injury. In addition to reviewing current knowledge, we present new aspects of the molecular structure of DAPk domains, and relate these findings to interacting partners of DAPk1, DAPk-regulation in NMDA-induced cerebral injury and novel approaches to blocking the injurious effects of DAPk1.

MRNA expression and hypermethylation of tumor suppressor genes apoptosis protease activating factor-1 and death-associated protein kinase in oral squamous cell carcinoma

Apoptosis protease activating factor-1 (Apaf-1) and death-associated protein kinase (DAPK) are p53 pathway-related genes that play significant roles in the activation of caspases, which are involved in mitochondrial-mediated apoptosis. The present study aimed to confirm the role of hyper-methylation of the Apaf-1 and DAPK gene promoter regions in oral squamous cell carcinoma (OSCC) and the effect of the demethylation drug, 5-aza-2′-deoxycytidine (DAC). mRNA from 53 OSCC samples, 23 normal oral mucosa samples and Tca8113 human tongue carcinoma cell lines was detected using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The DNA from each sample was analyzed using methylation-specific PCR (MSP). The Tca8113 cells were demethylated using DAC and the demethylation and re-expression of Apaf-1 and DAPK were analyzed. The Apaf-1 and DAPK mRNA expression index was decreased in 51 (96.23%) and 50 (94.34%) cases, respectively, in the tumor tissues. Hypermethylation of the Apaf-1 and DAPK promoter regions was detected in 46 (86.79%) and 38 (71.69%) cases, respectively. Promoter hypermethylation of the two genes correlated with a decreased mRNA expression in the tumor tissues. Subsequent to being treated with DAC, Apaf-1 and DAPK were demethylated and re-expressed in the Tca8113 cells. Apaf-1 and DAPK promoter hypermethylation may be associated with low gene expression in OSCC. Furthermore, a loss of Apaf-1 and DAPK expression may recover following demethylation. The data provide evidence that methylation exists in OSCC and may play a role in the development of this disease.