Silicon and Zinc Fertilizer Application Improves Grain Quality and Aroma in the japonica Rice Variety Nanjing 46.

This study examined how silicon and zinc fertilizers affect the quality and aroma of Nanjing 46. We applied nine different fertilizer treatments, one involving soil topdressing at the top fourth leaf-age stage and one involving foliar spraying during the booting stage of the silicon and zinc fertilizers. We tested the effects of the nine treatments on grain quality and aroma. Silicon and zinc fertilizers significantly affected the brown rice rate, milled rice rate, head rice rate, amylose content, gel consistency, RVA characteristic value, taste value, and aroma but did not affect the chalky grain rate, chalkiness, protein content, rice appearance, hardness, stickiness, balance, peak time, or pasting temperature. Silicon fertilizer decreased the rate of brown rice and milled rice, whereas zinc fertilizer increased the rate of brown rice and milled rice. Silicon and zinc fertilizers improved the head rice rate. Compared to silicon fertilizer, the impact of zinc fertilizer on increasing the head rice rate was more pronounced. Although the effects of silicon and zinc fertilizers on the amylose content and RVA characteristic value varied depending on the treatment, their application could lower the amylose content, increase gel consistency, improve breakdown viscosity, decrease setback viscosity, increase aroma, and improve the taste value of rice.

Circular RNA CircACAP2 regulates temporomandibular joint osteoarthritis via miR-21-5p/PLAG1 axis.

OBJECTIVES: The pathogenesis of temporomandibular joint osteoarthritis (TMJOA) remains not fully understood. Our previous studies demonstrated that miR-21-5p may participate in the TMJOA development and the interaction between circRNA-ACAP2 (CircACAP2) and miR-21-5p. Our present study aimed to explore the biological functions and regulatory mechanisms of CircACAP2 in TMJOA. MATERIALS AND METHODS: The differential expression pattern of CircACAP2 in OA and normal tissues or cells was detected. CircACAP2 biological functions experiments were performed in chondrocytes by overexpression and interference techniques. The interaction of CircACAP2 with miR-21-5p and downstream target mRNA, polymorphic adenoma gene 1 (PLAG1), was predicted by bioinformatic databases and then demonstrated by dual-luciferase reporter assay. The biological role of CircACAP2 in TMJOA was investigated and validated in a mouse model. RESULTS: The expression level of CircACAP2 was markedly reduced in OA cartilage and directly related to chondrocyte proliferation and apoptosis as well as ECM metabolism in the cartilage. CircACAP2 functioned in chondrocytes via targeting miR-21-5p and PLAG1. Overexpressing of CircACAP2 alleviated TMJOA in mouse models. CONCLUSIONS: The present study unveiled that CircACAP2/miR-21-5p/PLAG1 axis may play an important regulatory role in TMJOA progression, which may highlight a potentially effective intervention and therapeutic strategy for the treatment of TMJOA.

Prognostic value of association of OCT4 with LEF1 expression in esophageal squamous cell carcinoma and their impact on epithelial-mesenchymal transition, invasion, and migration.

Esophageal squamous cell carcinoma (ESCC) is a malignant disease with poor prognosis. Because of early metastasis prior to diagnosis and therapeutic resistance, ESCC has become one of the leading causes of cancer-related death. Here, we investigated the clinicopathological significance of the association of octamer-binding transcription factor 4 (OCT4) with lymphoid enhancer-binding factor 1 (LEF1) expression and the potential molecular mechanism in the epithelial-mesenchymal transition (EMT), invasion, and migration of ESCC. The expression of OCT4 and LEF1 was detected via immunohistochemistry analysis. High levels of LEF1 expression were observed in 95 ESCC specimens and were obviously associated with aberrant clinicopathological features and poor patient prognosis. Our previous study showed that OCT4 expression level is elevated in ESCC, and statistical analysis showed that the elevated expression of OCT4 and LEF1 in ESCC was significantly associated with histologic grade, lymph node metastasis, TNM stage, and poor patient prognosis. The specific inhibition of OCT4 expression via a lentivirus encoding OCT4-shRNA (LV-shOCT4) in Eca109 cells led to decreased levels of OCT4 and LEF1 in vitro. Additionally, we applied a rescue strategy by infecting LV-shOCT4 Eca109 cells with a LEF1 overexpression plasmid (p-LEF1) and detected changes in EMT, migration, and invasion. Unsurprisingly, the p-LEF1 group exhibited greater EMT, invasion, and migration than did the LV-shOCT4 and negative control groups. This study demonstrates for the first time the relationship between OCT4 and LEF1 expression. The combination of high expression of OCT4 and LEF1 was associated with clinicopathological features of atypical patients, and this combination might be an ideal prognostic factor in ESCC. OCT4 positively regulated LEF1 expression, and LEF1 mediated the effects of OCT4 in cancer cell EMT, invasion, and migration. The data presented here suggest that the inhibition of OCT4-LEF1 signaling may be a new therapeutic target for the treatment of ESCC.

Pim-1 Kinase Phosphorylates Cardiac Troponin I and Regulates Cardiac Myofilament Function.

BACKGROUND/AIMS: Pim-1 is a serine/threonine kinase that is highly expressed in the heart, and exerts potent cardiac protective effects through enhancing survival, proliferation, and regeneration of cardiomyocytes. Its myocardial specific substrates, however, remain unknown. In the present study, we aim to investigate whether Pim-1 modulates myofilament activity through phosphorylation of cardiac troponin I (cTnI), a key component in regulating myofilament function in the heart. METHODS: Coimmunoprecipitation and immunofluorescent assays were employed to investigate the interaction of Pim-1 with cTnI in cardiomyocytes. Biochemical, site directed mutagenesis, and mass spectrometric analyses were utilized to identify the phosphorylation sites of Pim1 in cTnI. Myofilament functional assay using skinned cardiac fiber was used to assess the effect of Pim1-mediated phosphorylation on cardiac myofilament activity. Lastly, the functional significance of Pim1-mediated cTnI in heart disease was determined in diabetic mice. RESULTS: We found that Pim-1 specifically interacts with cTnI in cardiomyocytes and this interaction leads to Pim1-mediated cTnI phosphorylation, predominantly at Ser23/24 and Ser150. Furthermore, our functional assay demonstrated that Pim-1 induces a robust phosphorylation of cTnI within the troponin complex, thus leading to a decreased Ca2+ sensitivity. Insulin-like growth factor 1 (IGF-1), a peptide growth factor that has been shown to stimulate myocardial contractility, markedly induces cTnI phosphorylation at Ser23/24 and Ser150 through increasing Pim-1 expression in cardiomyocytes. In a high-fat diabetic mice model, the expression of Pim1 in the heart is significantly decreased, which is accompanied by a decreased phosphorylation of cTnI at Ser23/24 and Ser150, further implicating the pathological significance of the Pim1/cTnI axis in the development of diabetic cardiomyopathy. CONCLUSION: Our results demonstrate that Pim-1 is a novel kinase that phosphorylates cTnI primarily at Ser23/24 and Ser150 in cardiomyocytes, which in turn may modulate myofilament function under a variety of physiological and pathophysiological conditions.

miR-214 inhibits invasion and migration via downregulating GALNT7 in esophageal squamous cell cancer.

Previous studies verified that miR-214 is of great significance in the invasion and migration of a variety of cancers. It has been demonstrated that UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7(GALNT7) is a putative target of miR-214. We performed this study to figure out how miR-214 and GALNT7 play their roles in the invasion and migration of esophageal squamous cell carcinoma (ESCC). The expression of miR-214 was significantly downregulated in tumors compared to the corresponding non-tumor tissues while GALNT7 showed an opposite tendency. The low expression of miR-214 and the high expression of GALNT7 were found positively correlated with poor tumor differentiation (P = 0.004), tumor invasion (P = 0.013), and lymph node metastasis (P = 0.012) in ESCC patients. Functional study demonstrated that overexpression of miR-214 or knockdown of GALNT7 could weaken invasive and migratory ability in Eca109, TE1, and KYSE150. Moreover, tumorigenicity assay showed us mice injected with cells containing miR-214 mimic or GALNT7 small interfering RNA formed substantially smaller tumors than that in miR-214 inhibitor group. Consequently, we concluded that miR-214 shows potential to be a diagnostic marker and therapeutic target in ESCC.

ANG Promotes Proliferation and Invasion of the Cell of Lung Squamous Carcinoma by Directly Up-Regulating HMGA2.

OBJECTIVE: To determine the mechanism of Angiogenin(ANG) function involved in the carcinogenesis of lung squamous cell carcinoma. METHODS: 12 patients' normal tissue and cancerous tissue were collected. ANG expression in the squamous cell carcinoma of the lung was evaluated by qRT-PCR and western-blot. The regulation of ANG on proliferation, migration, invasion, and apoptosis of SK-MES-1 cells were analyzed by Cell Counting Kit-8, Transwell migration chamber, Transwell invasion chamber, and Annexin V-FITC assay, respectively. PCR array was utilized for screening potential target genes of ANG. Chromatin immunoprecipitation(ChIP) assays and luciferase assay were adopted for investigation of ANG's direct regulation on HMGA2. RESULTS: ANG expression is increased in the squamous cell carcinoma of the lung tissue. In vitro experiments results indicated that overexpression of ANG promotes proliferation and invasion capability of SK-MES-1 cells. The candidate proliferation, migration, and invasion related ANG target gene found was HMGA2, expression levels of which were also enhanced in lung squamous cell carcinoma tissue. The direct regulation of ANG on HMGA2 was verified by ChIP and luciferase assay results. Furthermore, down-regulating HMGA2 significantly alleviated the suppression effects of ANG on proliferation, migration, and invasion of SK-MES-1 cells. CONCLUSIONS: Our data illustrated the mechanisms that ANG promoted the cell of SQCLC proliferation, migration, and invasion capacity via directly up-regulating HMGA2.

Pim1 kinase promotes angiogenesis through phosphorylation of endothelial nitric oxide synthase at Ser-633.

AIMS: Posttranslational modification, such as phosphorylation, plays an essential role in regulating activation of endothelial NO synthase (eNOS). In the present study, we aim to determine whether eNOS could be phosphorylated and regulated by a novel serine/threonine-protein kinase Pim1 in vascular endothelial cells (ECs). METHODS AND RESULTS: Using immunoprecipitation and protein kinase assays, we demonstrated that Pim1 specifically interacts with eNOS, which leads to a marked phosphorylation of eNOS at Ser-633 and increased production of nitric oxide (NO). Intriguingly, in response to VEGF stimulation, eNOS phosphorylation at Ser-633 exhibits two distinct phases: transient phosphorylation occurring between 0 and 60 min and sustained phosphorylation occurring between 2 and 24 h, which are mediated by the protein kinase A (PKA) and Pim1, respectively. Inhibiting Pim1 by either pharmacological inhibitor SMI-4a or the dominant-negative form of Pim1 markedly attenuates VEGF-induced tube formation, while Pim1 overexpression significantly increases EC tube formation and migration in an NO-dependent manner. Importantly, Pim1 expression and eNOS phosphorylation at Ser-633 were substantially decreased in high glucose-treated ECs and in the aorta of db/db diabetic mice. Increased Pim1 expression ameliorates impaired vascular angiogenesis in diabetic mice, as determined by an ex vivo aortic ring assay. CONCLUSION: Our findings demonstrate Pim1 as a novel kinase that is responsible for the phosphorylation of eNOS at Ser-633 and enhances EC sprouting of aortic rings from diabetic mice, suggesting that Pim1 could potentially serve as a novel therapeutic target for revascularization strategies.

Paenibacillin A, a new 2(1H)-pyrazinone ring-containing natural product from the endophytic bacterium Paenibacillus sp. Xy-2.

A new 2(1H)-pyrazinone ring-containing natural product, paenibacillin A (1), together with five known diketopiperazine derivatives 2-6 and two known isoflavones 7-8, was isolated from the culture of an endophytic bacterium Paenibacillus sp. Xy-2. The structure of compound 1 was elucidated by extensive spectral methods, including UV, IR, HR-ESI-MS, 1D and 2D NMR and ECD experiments. Compound 1 exhibited moderate cytotoxicity against HL-60 cell line with IC50 value of 50.48 µM.

Post-Transcriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Polypyrimidine Tract-Binding Protein 1.

OBJECTIVE: Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular function and its expression is regulated at post-transcriptional levels through a yet unknown mechanism. The purpose of this study is to elucidate the post-transcriptional factors regulating eNOS expression and function in endothelium. APPROACHES AND RESULTS: To elucidate the molecular basis of tumor necrosis factor (TNF)-α-mediated eNOS mRNA instability, biotinylated eNOS 3'-untranslational region (UTR) was used to purify its associated proteins by RNA affinity chromatography from cytosolic fractions of TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). We identified 2 cytosolic proteins, with molecular weight of 52 and 57 kDa, which specifically bind to eNOS 3'-UTR in response to TNF-α stimulation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis identified the 57-kDa protein as polypyrimidine tract-binding protein 1 (PTB1). RNA gel mobility shift and UV cross-linking assays demonstrated that PTB1 binds to a UCUU-rich sequence in eNOS 3'-UTR, and the C-terminal half of PTB1 is critical to this interaction. Importantly, PTB1 overexpression leads to decreased activity of luciferase gene fused with eNOS 3'-UTR as well as reduced eNOS expression and activity in human ECs. In HUVECs, we show that TNF-α markedly increased PTB1 expression, whereas adenovirus-mediated PTB1 overexpression decreased eNOS mRNA stability and reduced protein expression and endothelium-dependent relaxation. Furthermore, knockdown of PTB1 substantially attenuated TNF-α-induced destabilization of eNOS transcript and downregulation of eNOS expression. CONCLUSIONS: These results indicate that PTB1 is essential for regulating eNOS expression at post-transcriptional levels and suggest a novel therapeutic target for treatment of vascular diseases associated with inflammatory endothelial dysfunction.

Orphan Nuclear Receptor Nur77 Inhibits Cardiac Hypertrophic Response to Beta-Adrenergic Stimulation.

The orphan nuclear receptor Nur77 plays critical roles in cardiovascular diseases, and its expression is markedly induced in the heart after beta-adrenergic receptor (ß-AR) activation. However, the functional significance of Nur77 in ß-AR signaling in the heart remains unclear. By using Northern blot, Western blot, and immunofluorescent staining assays, we showed that Nur77 expression was markedly upregulated in cardiomyocytes in response to multiple hypertrophic stimuli, including isoproterenol (ISO), phenylephrine (PE), and endothelin-1 (ET-1). In a time- and dose-dependent manner, ISO increases Nur77 expression in the nuclei of cardiomyocytes. Overexpression of Nur77 markedly inhibited ISO-induced cardiac hypertrophy by inducing nuclear translocation of Nur77 in cardiomyocytes. Furthermore, cardiac overexpression of Nur77 by intramyocardial injection of Ad-Nur77 substantially inhibited cardiac hypertrophy and ameliorated cardiac dysfunction after chronic infusion of ISO in mice. Mechanistically, we demonstrated that Nur77 functionally interacts with NFATc3 and GATA4 and inhibits their transcriptional activities, which are critical for the development of cardiac hypertrophy. These results demonstrate for the first time that Nur77 is a novel negative regulator for the ß-AR-induced cardiac hypertrophy through inhibiting the NFATc3 and GATA4 transcriptional pathways. Targeting Nur77 may represent a potentially novel therapeutic strategy for preventing cardiac hypertrophy and heart failure.

Nonsense and missense mutation of mitochondrial ND6 gene promotes cell migration and invasion in human lung adenocarcinoma.

BACKGROUND: Previous study showed that mitochondrial ND6 (mitND6) gene missense mutation resulted in NADH dehydrogenase deficiency and was associated with tumor metastasis in several mouse tumor cell lines. In the present study, we investigated the possible role of mitND6 gene nonsense and missense mutations in the metastasis of human lung adenocarcinoma. METHODS: The presence of mitND6 gene mutations was screened by DNA sequencing of tumor tissues from 87 primary lung adenocarcinoma patients and the correlation of the mutations with the clinical features was analyzed. In addition, we constructed cytoplasmic hybrid cells with denucleared primary lung adenocarcinoma cell as the mitochondria donor and mitochondria depleted lung adenocarcinoma A549 cell as the nuclear donor. Using these cells, we studied the effects of mitND6 gene nonsense and missense mutations on cell migration and invasion through wounding healing and matrigel-coated transwell assay. The effects of mitND6 gene mutations on NADH dehydrogenase activity and ROS production were analyzed by spectrophotometry and flow cytometry. RESULTS: mitND6 gene nonsense and missense mutations were detected in 11 of 87 lung adenocarcinoma specimens and was correlated with the clinical features including age, pathological grade, tumor stage, lymph node metastasis and survival rate. Moreover, A549 cell containing mitND6 gene nonsense and missense mutation exhibited significantly lower activity of NADH dehydrogenase, higher level of ROS, higher capacity of cell migration and invasion, and higher pAKT and pERK1/ERK2 expression level than cells with the wild type mitND6 gene. In addition, NADH dehydrogenase inhibitor rotenone was found to significantly promote the migration and invasion of A549 cells. CONCLUSIONS: Our data suggest that mitND6 gene nonsense and missense mutation might promote cell migration and invasion in lung adenocarcinoma, probably by NADH dehydrogenase deficiency induced over-production of ROS.

KDM1A triggers androgen-induced miRNA transcription via H3K4me2 demethylation and DNA oxidation.

BACKGROUND: Androgen receptor (AR) is a ligand dependent transcription factor that regulates the transcription of target genes. AR activity is closely involved in the maintenance and progression of prostate cancer. After the binding with androgen, AR moves into nucleus and binds to DNA sequence containing androgen response elements (ARE). Flavin-dependent monoamine oxidase KDM1A is necessary for AR driven transcription while the mechanism remains unclear. METHODS: The association between androgen-dependent transcription and oxidation was tested through pharmaceutical inhibitions and siRNA knockdown of DNA oxidation repair components in prostate cancer cells. The recruitment of involved proteins and the histone methylation dynamics on ARE region was explored by chromatin immunoprecipitation (ChIP). RESULTS: Oxidation inhibition reduced AR dependent expression of KLK3, TMPRSS2, hsa-miR-125b2, and hsa-miR-133b. And such reduction could be restored by H2 O2 treatment. KDM1A recruitment and H3K4me2 demethylation on ARE regions, which produce H2 O2 , are associated with AR targets transcription. AR targets transcription and coupled oxidation recruit 8-oxoguanine-DNA glycosylase (OGG1) and the nuclease APEX1 to ARE regions. Such recruitment depends on KDM1A, and is necessary for AR targets transcription. CONCLUSION: Our work underlined the importance of histone demethylation and DNA oxidation/repairing machinery in androgen-dependent transcription. The present finds have implications for research into new druggable targets for prostate cancer relying on the cascade of AR activity regulation.

BRG1 overexpression in smooth muscle cells promotes the development of thoracic aortic dissection.

BACKGROUND: Here we investigated Brahma-related gene 1 (BRG1) expression in aortic smooth muscle cells (SMCs) and its role in the regulation of the pathological changes in aortic SMCs of thoracic arotic dissection (TAD). METHODS: BRG1, matrix metalloproteinase 2 (MMP2), and MMP9 mRNA and protein expression in human aortic specimens were examined by qPCR and western blot, respectively. The percentage of apoptotic and contractile SMCs in aortic specimens were determined by TUNEL assay and α-SMA immunohistochemical staining, respectively. The role of BRG1 in MMP2 and MMP9 expression, cell apoptosis, and phenotype transition in aortic SMCs were investigated using a human aortic SMC line via adenovirus mediated gene transfer. MMPs mRNA and protein levels were analyzed by qPCR and western blot, respectively. The percentage of apoptotic and contractile cells were determined through flow cytometry analysis. RESULTS: The expression level of BRG1 in the aortic walls (adventitia-removed) was significantly higher in the TAD than the normal group. BRG1 expression was positively correlated to expression of MMP2 and MMP9 and SMC apoptosis, but was negatively correlated to the percentage of contractile aortic SMCs in TAD specimens. In human aortic SMC line, BRG1 transfection led to significant upregulation of MMP2 and MMP9 expression and a concomitant increase in SMC apoptosis as well as a decrease in the percentage of contractile phenotype of cells. CONCLUSIONS: BRG1 is significantly upregulated in the aortic SMCs of TAD, and its overexpression might promote the development of TAD by increasing MMP2 and MMP9 expression, inducing SMC apoptosis and the transition from contractile to synthetic phenotype.

Intrathecal infusion of hydrogen-rich normal saline attenuates neuropathic pain via inhibition of activation of spinal astrocytes and microglia in rats.

BACKGROUND: Reactive oxygen and nitrogen species are key molecules that mediate neuropathic pain. Although hydrogen is an established antioxidant, its effect on chronic pain has not been characterized. This study was to investigate the efficacy and mechanisms of hydrogen-rich normal saline induced analgesia. METHODOLOGY/PRINCIPAL FINDINGS: In a rat model of neuropathic pain induced by L5 spinal nerve ligation (L5 SNL), intrathecal injection of hydrogen-rich normal saline relieved L5 SNL-induced mechanical allodynia and thermal hyperalgesia. Importantly, repeated administration of hydrogen-rich normal saline did not lead to tolerance. Preemptive treatment with hydrogen-rich normal saline prevented development of neuropathic pain behavior. Immunofluorochrome analysis revealed that hydrogen-rich normal saline treatment significantly attenuated L5 SNL-induced increase of 8-hydroxyguanosine immunoreactive cells in the ipsilateral spinal dorsal horn. Western blot analysis of SDS/PAGE-fractionated tyrosine-nitrated proteins showed that L5 SNL led to increased expression of tyrosine-nitrated Mn-containing superoxide dismutase (MnSOD) in the spinal cord, and hydrogen-rich normal saline administration reversed the tyrosine-nitrated MnSOD overexpression. We also showed that the analgesic effect of hydrogen-rich normal saline was associated with decreased activation of astrocytes and microglia, attenuated expression of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the spinal cord. CONCLUSION/SIGNIFICANCE: Intrathecal injection of hydrogen-rich normal saline produced analgesic effect in neuropathic rat. Hydrogen-rich normal saline-induced analgesia in neuropathic rats is mediated by reducing the activation of spinal astrocytes and microglia, which is induced by overproduction of hydroxyl and peroxynitrite.

Structure determination of two new indole-diterpenoids from Penicillium sp. CM-7 by NMR spectroscopy.

Two new indole-diterpenoids 4b-deoxy-1'-O-acetylpaxilline (1) and 4b-deoxypenijanthine A (2) were isolated from the fermentation broth and the mycelia of the soil fungus Penicillium sp. CM-7, along with three known structurally related compounds, 1'-O-acetylpaxilline (3), paspaline (4) and 3-deoxo-4b-deoxypaxilline (5). The structures of compounds 1 and 2 were elucidated by extensive spectroscopic methods, especially 2D NMR, and their absolute configurations were suggested on the basis of the circular dichroism spectral analysis and the NOESY data.

Proteinase-activated receptor 1 contributed to up-regulation of enkephalin in keratinocytes of patients with obstructive jaundice.

BACKGROUND: Skin synthesis of endogenous opioids such as enkephalin is considered to be increased in cholestatic rodents, which may induce antinociception in cholestatic liver disease. No studies have reported yet the expression of skin enkephalin in patients with cholestasis. METHODS: Electrical pain threshold, postoperative morphine consumption, and skin enkephalin expression were measured in patients with jaundice (n = 18) and control patients (n = 16). Male Sprague-Dawley rats (n = 52) and human keratinocyte cell line HaCaT were used in vivo and in vitro studies, respectively. Nociceptive thresholds and plasma and skin levels of methionine-enkephalin were compared in protease-activated receptors-1-antagonized and control bile duct-ligated rats. In in vitro study, the effect on thrombin-induced enkephalin expression was examined and the role of extracellular regulated protein kinases 1/2 and p38 was investigated. RESULTS: The authors found that: (1) the electrical pain threshold (mean ± SD) was 1.1 ± 0.1 mA in control patients, whereas it was significantly increased in patients with jaundice (1.7 ± 0.3 mA); 48-h postoperative morphine consumption was approximately 50% higher in the control group than that in the group with jaundice; (2) Skin keratinocytes enkephalin expression was increased in the patients with jaundice; (3) Protease-activated receptors-1 antagonist 1 µg·kg(-1)·day(-1) treatment to the bile duct-ligated rats significantly reduced plasma levels of methionine-enkephalin, nociceptive thresholds, and keratinocytes enkephalin expression; and (4) protease-activated receptors-1 activation induced enkephalin expression through phosphorylation of extracellular regulated protein kinases 1/2 and p38 in keratinocytes. CONCLUSION: Protease-activated receptors-1 activation in peripheral keratinocytes may play an important role in the local synthesis of enkephalin during cholestasis.

Protein-L-isoaspartate (D-aspartate) O-methyltransferase protects cardiomyocytes against hypoxia induced apoptosis through inhibiting proapoptotic kinase Mst1.

BACKGROUND: Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Hippo kinase from Drosophila and it is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart is not known. METHODS AND RESULTS: To identify novel cardiac proteins that may regulate Mst1 activity in the heart under pathophysiological conditions, a yeast two-hybrid screening of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait was performed. As a result, protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) was identified as an Mst1-interacting protein. The interaction of PCMT1 with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiomyocytes, in which PCMT1 interacted with the kinase domain of Mst1, but not with its C-terminal regulatory domain. Overexpression of PCMT1 did not affect the Mst1 expression, but significantly attenuated the Mst1 activation and its apoptotic effects in response to the hypoxia/reoxygenation induced injury in cardiomyocytes. Indeed, upregulation of PCMT1 by CGP3466B, a compound related to the anti-Parkinson's drug R-(-)-deprenyl with potent antiapoptotic effects, inhibited the hypoxia/reoxygenation induced Mst1 activation and cardiomyocyte apoptosis. CONCLUSIONS: These findings implicate PCMT1 as a novel inhibitor of Mst1 activation in cardiomyocytes and suggest that targeting PCMT1 may prevent myocardial apoptosis through inhibition of Mst1.

Glyceraldehyde-3-phosphate dehydrogenase interacts with proapoptotic kinase mst1 to promote cardiomyocyte apoptosis.

Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease.