Down-regulation Of Protein Kinase Research Articles

Lysophosphatidic Acid Prevents Ischemia Reperfusion Injury but does not Prevent Tubular Dysfunction

Lysophosphatidic acid (LPA) protects the kidneys from tissue ischemic reperfusion injury (IRI), but its impact on renal function is primarily limited to glomerular function. We estimated the status of renal function by a complete glomerular and tubular functional analysis to test the hypothesis that LPA treatment during ischemia-reperfusion (I/R) protects renal function by attenuating IRI. Male Wistar rats were subjected to bilateral kidney I/R. Along with ischemia, LPA was administered. LPA increased levels of plasma LPA, downregulated LPA2R, prevented interstitial fibronectin and TGF-β1 accumulation, and prevented a decrease in glomerular filtration rate (GFR). I/R increased urine volume and proteinuria and decreased fractional Na+ excretion (FENa) and urine osmolality. These effects were not prevented by LPA. The reduction in FENa was attributed to disruption in tubular Na+ transport and downregulation of protein kinase C (PKC) activity. LPA treatment maintained (Na++K+)ATPase activity to the control level, due to a sustained sensitivity to PLC/PKC pathway. Na+-ATPase activity was insensitivity to LPA treatment. This ineffectiveness was associated with downregulation of LPA2R, resulting in low FENa. Altogether, LPA treatment maintained normal kidney structure and prevented the reduction of glomerular function. However, even in the setting of preserved GFR, impaired tubular function may present a high risk for silent progression of kidney disease.

Phoenix dactylifera L. Seed Extract Exhibits Antioxidant Effects and Attenuates Melanogenesis in B16F10 Murine Melanoma Cells by Downregulating PKA Signaling.

Background: The mode of action of Phoenix dactylifera seed extract in skin care has never been explored. Methods: P. dactylifera L. seeds were extracted by ultrasonic extraction. The antioxidant characteristics of the extract were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS+) assays and scavenging methods. The total phenolic content, reducing capacity, iron (II) ion-chelation, and intracellular reactive oxygen species (ROS)-scavenging capacities were also investigated. The effects of P. dactylifera L. seed extract on melanogenesis were evaluated spectrophotometrically by a mushroom tyrosinase activity assay, determination of intracellular tyrosinase activity, and melanin content. The expression levels of melanogenesis-related proteins were analyzed by Western blotting. Results: The results revealed that the P. dactylifera L. seed extract exerted apparent antioxidant capacity and significantly decreased intracellular ROS content at concentrations of 0.245 and 0.49 (mg/mL). Furthermore, the extract decreased the expression of melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and tyrosinase-related protein-2 (TRP2), and inhibited melanogenesis in B16F10 cells. Conclusions: Our results revealed that P. dactylifera L. seed extract attenuated melanogenesis in B16F10 cells by downregulating protein kinase A (PKA) signaling pathways. Hence, the extract could be used as a type of skin-whitening agent in skin care products.

Curcumin reverses diabetic nephropathy in streptozotocin-induced diabetes in rats by inhibition of PKCβ/p66Shc axis and activation of FOXO-3a

This study investigated if the nephroprotective effect of Curcumin in streptozotocin-induced type 1 diabetes mellitus (DM) in rats involves downregulation/inhibition of p66Shc and examined the underlying mechanisms. Rats were divided into 4 groups (n = 12/group) as control, control + Curcumin (100 mg/kg), T1DM, and T1DM + Curcumin. Curcumin was administered orally to control or diabetic rats for 12 weeks daily. As compared to diabetic rats, Curcumin didn't affect either plasma glucose or insulin levels but significantly reduced serum levels of urea, blood urea nitrogen, and creatinine, and concurrently reduced albumin/protein urea and increased creatinine clearance. It also prevented the damage in renal tubules and mitochondria, mesangial cell expansion, the thickness of the basement membrane. Mechanistically, Curcumin reduced mRNA and protein levels of collagen I/III and transforming growth factor- β-1 (TGF-β1), reduced inflammatory cytokines levels, improved markers of mitochondrial function, and suppressed the release of cytochrome-c and the activation of caspase-3. In the kidneys of both control and diabetic rats, Curcumin reduced the levels of reactive oxygen species (ROS), increased mRNA levels of manganese superoxide dismutase (MnSOD) and gamma-glutamyl ligase, increased glutathione (GSH) and protein levels of Bcl-2 and MnSOD, and increased the nuclear levels of nuclear factor2 (Nrf2) and FOXO-3a. Besides, Curcumin reduced the nuclear activity of the nuclear factor-kappa B (NF-κB), downregulated protein kinase CβII (PKCβII), NADPH oxidase, and p66Shc, and decreased the activation of p66Shc. In conclusion, Curcumin prevents kidney damage in diabetic rats by activating Nrf2, inhibiting Nf-κB, suppressing NADPH oxidase, and downregulating/inhibiting PKCβII/p66Shc axis.

84-OR: Laminin-Islet Interactions Protect against Cytokine-Mediated Beta-Cell Death via Down-Regulation of Protein Kinase C Delta

Islets are surrounded by a specialized extracellular matrix (ECM). Interactions with the peri-islet ECM, specifically laminin, promote islet viability and function. In T1D during immune infiltration the peri-islet ECM is broken down and β-cell death occurs in part through production of high levels of Th1 cytokines. Recent work has shown that cytokines alter islet function via protein kinase C Δ (PKCΔ) dependent mechanisms and that islet interactions with laminin reduce PKCΔ activity. We hypothesize that laminin-islet interactions protect against cytokine-mediated β-cell death via downregulation of PKCΔ. To test our hypothesis, we used a 3D reverse thermal gel (RTG) scaffold with either laminin-10 (RTG-Lam) or lysine (RTG-Lys) as a control. Islets were isolated from WT or mice with an inducible β-cell specific PKCΔ knockout (PKCΔ-βKO) and encapsulated in RTG-Lam and RTG-Lys for 24hrs with or without cytokines (10ng/mL TNF-α, 5ng/mL IL-1β, 100ng/mL IFN-γ). Viability, PKCΔ translocation, and PKCΔ activity were measured in encapsulated islets in situ using fluorescent biosensors or by western blot. PKCΔ-βKO islets were protected against cytokine-induced death compared to controls (p=0.003). Cytokines increased β-cell death in RTG-Lys (p<0.001), while islets encapsulated in RTG-Lam were protected against cytokine-mediated death (p<0.04). Islets encapsulated in RTG-Lam had reduced PKCΔ activity at the cell membrane compared to RTG-Lys and were also protected against cytokine-mediated increases in PKCΔ activity at the cell membrane. In summary, islet interactions with laminin protect against cytokine-induced β-cell death via downregulation of PKCΔ activity at the cell membrane. Our results suggest that loss of islet interactions with laminin, as occurs in T1D, may make islets more susceptible to cytokine-mediated death. This mechanism of β-cell death provides a novel therapeutic target to prevent β-cell death and halt the progression of T1D. Disclosure N.L. Farnsworth: None. R.A. Piscopio: None. R.K. Benninger: None. Funding JDRF (3-APF-2019-749-A-N, 1-FAC-2020-891-A-N); Colorado Clinical and Translational Sciences Institute (CO-M-19-133)

Isothermal kinase-triggered supramolecular assemblies as drug sensitizers.

Protein kinases, the main regulators of a vast map of cellular processes, are the most attractive targets in drug discovery. Despite a few successful examples of protein kinase inhibitors, the drug discovery strategy of downregulating protein kinase activity has been quite limited and often fails even in animal models. Here, we utilize protein kinase A (PKA) activity to design PKA-triggered supramolecular assemblies with anticancer activities. Grafting a suitable peptide to PNIPAM raises the critical temperature of the LCST polymer above body temperature. Interestingly, the corresponding phosphorylated polymer has a critical temperature below body temperature, making this peptide-appended PNIPAM a suitable polymer for the PKA-triggered supramolecular assembly process. PKA-triggered assembly occurs selectively in PKA-upregulated MCF-7 cells, which disturbs the cytoskeleton and sensitizes cancer cells against doxorubicin. The chemosensitization is also observed in vivo to identify effective tumor inhibitors with satisfactory biocompatibility. Overall, this phosphorylation-induced (in principle, PKA-catalyzed) supramolecular assembly opens up a promising chemotherapy strategy for combating kinase-upregulated cancer.

6mer Seed Toxicity in Viral microRNAs.

SummaryMicroRNAs (miRNAs) are short double-stranded noncoding RNAs (19-23 nucleotides) that regulate gene expression by suppressing mRNAs through RNA interference. Targeting is determined by the seed sequence (position 2-7/8) of the mature miRNA. A minimal G-rich seed of just six nucleotides is highly toxic to cells by targeting genes essential for cell survival. A screen of 215 miRNAs encoded by 17 human pathogenic viruses (v-miRNAs) now suggests that a number of v-miRNAs can kill cells through a G-rich 6mer sequence embedded in their seed. Specifically, we demonstrate that miR-K12-6-5p, an oncoviral mimic of the tumor suppressive miR-15/16 family encoded by human Kaposi sarcoma-associated herpes virus, harbors a noncanonical toxic 6mer seed (position 3-8) and that v-miRNAs are more likely than cellular miRNAs to utilize a noncanonical 6mer seed. Our data suggest that during evolution viruses evolved to use 6mer seed toxicity to kill cells.

Women with polycystic ovary syndrome present with altered endometrial expression of stanniocalcin-1†.

Stanniocalcin-1 (STC-1) is a pro-survival factor that protects tissues against stressors, such as hypoxia and inflammation. STC-1 is co-expressed with the endometrial receptivity markers, and recently endometrial STC-1 was reported to be dysregulated in endometriosis, a condition linked with endometrial progesterone resistance and inflammation. These features are also common in the endometrium in women with polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. Given that women with PCOS present with subfertility, pregnancy complications, and increased risk for endometrial cancer, we investigated endometrial STC-1 expression in affected women. Endometrial biopsy samples were obtained from women with PCOS and controls, including samples from overweight/obese women with PCOS before and after a 3-month lifestyle intervention. A total of 98 PCOS and 85 control samples were used in immunohistochemistry, reverse-transcription polymerase chain reaction, or in vitro cell culture. STC-1 expression was analyzed at different cycle phases and in endometrial stromal cells (eSCs) after steroid hormone exposure. The eSCs were also challenged with 8-bromo-cAMP and hypoxia for STC-1 expression. The findings indicate that STC-1 expression is not steroid hormone mediated although secretory-phase STC-1 expression was blunted in PCOS. Lower expression seems to be related to attenuated STC-1 response to stressors in PCOS eSCs, shown as downregulation of protein kinase A activity. The 3-month lifestyle intervention did not restore STC-1 expression in PCOS endometrium. More studies are warranted to further elucidate the mechanisms behind the altered endometrial STC-1 expression and rescue mechanism in the PCOS endometrium.

The Interplay between Maternal and Post-Weaning High-Fat Diet and Gut Microbiota in the Developmental Programming of Hypertension.

Excessive intake of saturated fat has been linked to hypertension. Gut microbiota and their metabolites, short-chain fatty acids (SCFAs), are known to be involved in the development of hypertension. We examined whether maternal and post-weaning high-fat (HF) diet-induced hypertension in adult male offspring is related to alterations of gut microbiota, mediation of SCFAs and their receptors, and downregulation of nutrient-sensing signals. Female Sprague–Dawley rats received either a normal diet (ND) or HF diet (D12331, Research Diets) during pregnancy and lactation. Male offspring were put on either the ND or HF diet from weaning to 16 weeks of age, and designated to four groups (maternal diet/post-weaning diet; n = 8/group): ND/ND, HF/ND, ND/HF, and HF/HF. Rats were sacrificed at 16 weeks of age. Combined HF/HF diets induced elevated blood pressure (BP) and increased body weight and kidney damage in male adult offspring. The rise in BP is related to a downregulated AMP-activated protein kinase (AMPK)–peroxisome proliferator-activated receptor co-activator 1α (PGC-1α) pathway. Additionally, HF/HF diets decreased fecal concentrations of propionate and butyrate and decreased G protein-coupled receptor 41 (GPR41), but increased olfactory receptor 78 (Oflr78) expression. Maternal HF diet has differential programming effects on the offspring’s microbiota at 3 and 16 weeks of age. Combined HF/HF diet induced BP elevation was associated with an increased Firmicutes to Bacteroidetes ratio, increased abundance of genus Akkermansia and phylum Verrucomicrobia, and reduced abundance in genus Lactobacillus. Maternal gut microbiota-targeted dietary interventions might be reprogramming strategies to protect against programmed hypertension in children and their mothers on consumption of a fat-rich diet.

β-Cryptoxanthin induced anti-proliferation and apoptosis by G0/G1 arrest and AMPK signal inactivation in gastric cancer

β-Cryptoxanthin has been associated with reduced-risk of some cancers. However, the mechanisms of β-cryptoxanthin still remain unclearly understood in gastric cancer (GC). In this study, we examined the effect of β-cryptoxanthin on AMPK signal in human gastric cancer cells. AGS and SGC-7901 cells were treated with β-cryptoxanthin (0-40 μM) and AGS cells were injected in BALB/c (nu/nu) mice to analyze the effect of β-cryptoxanthin on GC. We found that β-cryptoxanthin induced inhibitory effect on the cell viability in a time- and concentration-dependent manner. The number of migrated cells and protein levels of matrix metalloproteinase (MMP) −2 and MMP-9 were obviously decreased. β-Cryptoxanthin treatment induced G0/G1 arrest, and reduced the expression of Cyclin E, Cyclin D1, cyclin-dependent kinases (CDK) of CDK4 and CDK6, and increased the expression of p53 and p21 in the two GC cells. Additionally, β-cryptoxanthin induced apoptosis and increased the expression of cleaved caspase-3, -8, -9 as well as cytochrome C (cyt C). β-Cryptoxanthin induced AMP-activated protein kinase (AMPK) signal inactivation by the down-regulation of protein kinase A (PKA), p-AMPK, eukaryotic elongation factor 2 kinase (eEF2k). Furthermore, β-cryptoxanthin inhibited tumor growth through suppressing the tumor volume and weight, inducing apoptotic cells. Besides, β-cryptoxanthin induced significant reductions of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). In conclusion, our data provide the novel evidence to understand the mechanism of anti-pcancer of β-cryptoxanthin and indicate that β-cryptoxanthin can serve as a promising chemopreventive agent against gastric cancer.

Downregulation of A2AR by siRNA loaded PEG-chitosan-lactate nanoparticles restores the T cell mediated anti-tumor responses through blockage of PKA/CREB signaling pathway

Adenosine and its receptors are novel promising targets for cancer immunotherapy. In here, we aimed to evaluate the efficacy of Polyethylene glycol (PEG)-chitosan-lactate (PCL) nanoparticles (NPs) loaded with A2AR-specific siRNA for interfering with differentiation and function of T cells derived from the 4T1 breast tumor-bearing Balb/C mice, ex vivo. The size of synthesized NPs was about 100 nm in association with low polydispersive index (pdi < 0.3) and a zeta potential of 11 mV. In association with good physicochemical characteristics, NPs exhibited high transfection efficiency in T cells and low toxicity on the various cell lines. T cells were treated with A2AR siRNA-loaded NPs demonstrated suppressed expression of A2AR which was associated with increased proliferation, reduced apoptosis, increased production of inflammatory and reduced secretion of inhibitory cytokines compared to untreated T cells. Moreover, differentiation of conventional T cells purified from tumor-bearing mice to regulatory T cells (Treg) was blocked using A2AR-specific siRNA-loaded NPs. These immune-stimulatory effects were in part through downregulation of protein kinase A/cAMP-response element binding protein (PKA/CREB) axis and upregulation of nuclear factor-κB (NF-κB).

Salvia miltiorrhiza Restrains Reactive Oxygen Species-Associated Pulmonary Fibrosis via Targeting Nrf2-Nox4 Redox Balance.

Pulmonary fibrosis (PF) is characterized by myofibroblast activation, which can be triggered by oxidative stress. In this study, we investigated the antifibrotic effect of the ethyl acetate extract of Salvia miltiorrhiza (EASM) on PF and examined the underlying molecular mechanism. EASM suppressed myofibroblast activation with reduced extracellular matrix deposition in the lungs of mice subjected to bleomycin (BLM) challenge, demonstrating the inhibitory effects on PF. EASM positively alleviated oxidative stress by upregulating nuclear factor-erythroid 2-related factor 2 (Nrf2) and concomitantly downregulating NADPH oxidase 4 (Nox4) in the lungs of BLM-treated mice. This effect was also observed in an in vitro model of transforming growth factor beta 1 (TGF-1)-stimulated fibroblast activation. EASM reduced reactive oxygen species (ROS) generation in fibroblasts by stabilizing Nrf2 protein with promoting kelch-like ECH-associated protein 1 (Keap1) degradation. Nrf2 knockdown in the lungs of BLM-treated mice diminished the inhibitory effects of EASM on fibrosis, providing evidence in vivo to address the unique role of Nrf2. Additionally, EASM inhibited TGF-1/Smad3 signaling by downregulating protein kinase C delta (PKC- and Smad3 phosphorylation (p-Smad3), which led to suppression of the TGF-1-induced fibrogenic response. These results indicate that EASM exhibits potent antifibrotic activity in vitro and in vivo, which might be associated with activation of Nrf2 pathway and inhibition of TGF-1/Smad3 pathway. Our findings support that EASM may act as an effective antifibrotic remedy for PF.

Antioxidative effects of complementary therapy with Salvia miltiorrhiza in ischemic heart disease

Background: Salvia miltiorrhiza (SM) is a Chinese herb widely used for ischemic heart diseases (IHD), yet little is known about the cellular mechanisms. The aims of this study were to investigate mechanisms of SM. Methods: The rat A10 cells line, a vascular smooth muscle cells line isolated from rat thoracic embryonic aorta was as a study model. The SM roots aqueous extract , MTT assay, cytotoxicity assay, two-dimensional electrophoresis coupled with MALDI-TOF mass spectrometry , western blot analysis, and biological network analysis were applied for the elucidation of protein changes characterizing the response of the rat A10 cells into the homocysteine(Hcy) -induced oxidative stress. Results: Our study showed that a low dose (0.015 mg/mL) of the SM significantly inhibited growth (>60%, p < 0.05) of the Hcy stimulated rat A10 cells. In addition, concentration of intracellular reactive oxygen species obviously decreased in the rat A10 cells after its incubation with SM in terms of catalase increasing activity. Next, marked down-regulation of protein kinase C beta-1 and phosphorylated mitogen-activated protein kinase expression suggest that observed inhibitory effect of the SM on the Hcy-induced growth of rat A10 cells was realized via the PKC/p44/42 MAPK- dependent pathway. The intensity changes of 10 protein spots in response of the rat A10 cells to the Hcy-induced oxidative damage as alpha-4-tropomyosin, vimentin, F1F0- ATP synthase (beta subunit), glucose regulated protein 75, actin (fragment), prohibitin, capping protein, plakoglobin, endoplasmic reticulum protein 29, and peptidylprolyl isomerase A, were detected with statistical significance (p < 0.05). Meanwhile, it was showed that used here SM resist carbonylation of vimentin, alpha-4-tropomyosin and GRP75, respectively, leading to phenotype transformations in the rat A10 cells. Conclusion: These data suggest that SM may exert its protective effect in IHD through circulating ROS suppression and subsequent modulation of protein carbonylation in rat aortic smooth muscle cells. Biography Yu-Chiang Hung grew up in Taiwan and received his Bachelor's degree from the China Medical University in 1990 , another M.D . degree from the National Yang-Ming University in 1992 , and Ph.D. degree from the Chang Gung University in 2010. His PhD thesis was “Functional proteomic study in bioactive compounds of radix Salvia miltiorrhiza”. Dr. Hung had found that Salvia miltiorrhiza could inhibit vascular smooth muscle cell proliferation to treat atherosclerosis. He is an attending and director of Department of Chinese medicine in Kaohsiung Chang Gung Memorial Hospital now. His specialty is Chinese medicine , cardiovascular diseases ,Chinese herbs , and acupuncture. He has published 39 papers in SCI journals and has been serving as an editorial board member of repute.

Anticancer effect of Dracaena arborea leaf extract through down-regulation of VCAM-1 and EMT proteins via suppressing Akt/PKC pathway

Dracaena arborea (Willd.) Link is known to be used in traditional medicine for the treatment of various dis-eases. Because it possesses steroidal saponins and flavonoids which display a variety of biological actions including anti-car-cinogenic, it might exhibit anti-cancer effect. However, the anti-cancer effect of D. arborea is not well known. Therefore, in this study, we investigated anti-cancer effect of D. arborea on highly metastatic human breast cancer cells MDA-MB-231. Methanolic extract of leaves of Dracaena arborea (MEDA) decreased cell viability of MDA-MB-231 cells, but not endothelial cells (ECs), in a dose-dependent manner for 24 h. Then, pretreatment of MEDA significantly inhibited the binding of MDA-MB-231 to ECs and the invasion of MDA-MB-231 in the presence of tumor necrosis factor (TNF)-α or not. Pretreatment of MEDA significantly inhibited vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression both in TNF-α-treated MDA-MB-231 cells and ECs at the indicated doses, and the inhibitory effect was more prominent in VCAM-1 rather than ICAM-1. TNF-α-mediated induction of β-catenin and Snail-1 and secretion of matrix metalloproteinase-9 (MMP-9) were significantly down-regulated by MEDA from 50 μg/ml and 100 μg/ml, respectively. Fur-thermore, MEDA effectively down-regulated protein kinase C (PKC) and Akt activation by TNF-α, suggesting that inhi-bition of PKC and Akt pathways by MEDA are responsible for differential inhibition of VCAM-1 and the epithelial&#8211; mesenchymal transition (EMT)-related proteins β-catenin and Snail-1. Taken together, MEDA exhibits anti-cancer effects through inhibition of adhesion of cancer cells to ECs and the invasion of cancer cells through down-regulation of VCAM-1 and EMT proteins via suppressing Akt/PKC pathway, suggesting a possibility to be served as a therapeutic agent against cancer metastasis.

O-GLCNACYLATION MODULATES PKA-CREB SIGNALING IN A MANNER SPECIFIC TO PKA CATALYTIC SUBUNIT ISOFORMS

Down-regulation of Protein kinase A (PKA)- cAMP response element binding protein (CREB) signaling is believed to be responsible for the learning and memory deficit in Alzheimer's disease (AD). It is known that isoforms a and b of PKA catalytic subunit (PKAc) and CREB are post-translationally modified with O-GlcNAc. OGT or OGA were overexpressed in HEK-293FT cells to examine the role of O-GlcNAcylation in CREB signaling. PKAc isoforms were co-transfected with OGT or OGA in N2a cells, and the effects of PKAc isoforms on O-GlcNAcylation and CREB signaling were identified. OGT was overexpressed or knocked down together with insulin treatment in HEK-293FT cells. O-GlcNAcylation and CREB signaling were checked by western blot. We found that up-regulation of O-GlcNAcylation enhanced CREB phosphorylation, but suppressed CREB expression in exogenous PKAc isoform-unspecific manner. PKAc isoforms affected exogenous expression of OGT or OGA and protein O-GlcNAcylation differently. Up-regulation of O-GlcNAcylation did not significantly affect net PKAca-CREB signaling, but enhanced PKAcb-CREB signaling. The role of O-GlcNAcylation in PKA-CREB signaling was desensitized by insulin treatment. We determined the role of O-GlcNAcylation in PKAc isoform-specific CREB signaling. Our data suggests O-GlcNAcylation modulates PKA-CREB signaling by regulating expression and phosphorylation of CREB in a PKAc isoform-specific manner.

Imoxin attenuates high fructose-induced oxidative stress and apoptosis in renal epithelial cells via downregulation of protein kinase R pathway.

Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a ubiquitously expressed serine/threonine kinase, is a key inducer of inflammation, insulin resistance, and glucose homeostasis in obesity. Recent studies have demonstrated that PKR can respond to metabolic stress in mice as well as in humans. However, the underlying molecular mechanism is not fully understood. The aim of this study was to examine the effect of high fructose (HF) in cultured renal tubular epithelial cells (NRK-52E) derived from rat kidney and to investigate whether inhibition of PKR could prevent any deleterious effects of HF in these cells. PKR expression was determined by immunofluorescence staining and Western blotting. Oxidative damage and apoptosis were measured by flow cytometry. HF-treated renal cells developed a significant increase in PKR expression. A significant increase in reactive oxygen species generation and apoptosis was also observed in HF-treated cultured renal epithelial cells. All these effects of HF were attenuated by a selective PKR inhibitor, imoxin (C16). In conclusion, our study demonstrates PKR induces oxidative stress and apoptosis, is a significant contributor involved in vascular complications and is a possible mediator of HF-induced hypertension. Inhibition of PKR pathway can be used as a therapeutic strategy for the treatment of cardiovascular and metabolic disorders.

Fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C

BackgroundChanges in metabolic pathway preferences are key events in the reprogramming process of somatic cells to induced pluripotent stem cells (iPSCs). The optimization of metabolic conditions can enhance reprogramming; however, the detailed underlying mechanisms are largely unclear. By comparing the gene expression profiles of somatic cells, intermediate-phase cells, and iPSCs, we found that carnitine palmitoyltransferase (Cpt)1b, a rate-limiting enzyme in fatty acid oxidation, was significantly upregulated in the early stage of the reprogramming process.MethodsMouse embryonic fibroblasts isolated from transgenic mice carrying doxycycline (Dox)-inducible Yamanaka factor constructs were used for reprogramming. Various fatty acid oxidation-related metabolites were added during the reprogramming process. Colony counting and fluorescence-activated cell sorting (FACS) were used to calculate reprogramming efficiency. Fatty acid oxidation-related metabolites were measured by liquid chromatography–mass spectrometry. Seahorse was used to measure the level of oxidative phosphorylation.ResultsWe found that overexpression of cpt1b enhanced reprogramming efficiency. Furthermore, palmitoylcarnitine or acetyl-CoA, the primary and final products of Cpt1-mediated fatty acid oxidation, also promoted reprogramming. In the early reprogramming process, fatty acid oxidation upregulated oxidative phosphorylation and downregulated protein kinase C activity. Inhibition of protein kinase C also promoted reprogramming.ConclusionWe demonstrated that fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C activity in the early stage of the reprogramming process. This study reveals that fatty acid oxidation is crucial for the reprogramming efficiency.

Eicosapentaenoic acid reduces indoleamine 2,3-dioxygenase 1 expression in tumor cells.

Marine plants and animals have omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). EPA is required for biological processes, but humans are unable to synthesize them and must be obtained from dietary sources. EPA has been used as an antitumor agent but the molecular mechanisms for the regulation of tumor microenvironment immunity by EPA are still unknown. The indoleamine 2,3-dioxygenase 1 (IDO) catalyzes conversion of tryptophan to kynurenine to induce immune evasion in tumor microenvironment. In this study, EPA inhibited the expression of IDO via downregulation of protein kinase B (Akt)/mammalian targets of rapamycin (mTOR) signaling pathway in tumor cells. Meanwhile, a significant decrease in kynurenine levels and increase in T cell survival were observed after tumor cells treated with EPA. The results demonstrated that EPA can activate host antitumor immunity by inhibiting tumor IDO expression. Therefore, our finding suggests that EPA can be enormous potential for cancer immunotherapy.

Aspirin ameliorates cerebral infarction through regulation of TLR4/NF‑κB‑mediated endoplasmic reticulum stress in mouse model

Cerebral infarction is a cerebrovascular disease caused by local brain ischemic necrosis or softening, which is associated with diabetes, obesity, hypertension and rheumatic heart arrhythmia. Previous studies have indicated that aspirin is a potential oral anticoagulant in the treatment of cerebral ischemic stroke. However, the potential mechanism mediated by aspirin in cerebral infarction therapy is not well understood. The present study analyzed the therapeutic effects of aspirin on cerebral infarction and investigated the underlying molecular mechanism of aspirin‑ameliorated benefits for thrombolysis. The results demonstrated that aspirin inhibited inflammation and apoptosis of cerebrovascular endothelial cells in a mouse model of cerebral infarction. Aspirin treatment suppressed toll‑like receptor (TLR)4 and nuclear factor (NF)‑κB expression in cerebrovascular endothelial cells. Endoplasmic reticulum (ER) stress was suppressed by aspirin treatment through the downregulation of protein kinase R‑like endoplasmic reticulum kinase, eukaryotic translation initiation factor 2 subunit 1 and C/EBP homologous protein expression levels in cerebrovascular endothelial cells. It was identified that knockdown of TLR4 inhibited aspirin‑mediated downregulation of NF‑κB signaling pathway and ER stress in cerebrovascular endothelial cells. Expression levels of adenosine diphosphate plasminogen activator inhibitors, von Willebrand factor and thromboxane were downregulated in cerebrovascular endothelial cells and in serum in experimental mice. The results demonstrated that aspirin was beneficial forthrombolysis by decreasing thrombin‑activatable fibrinolysis inhibitor and plasminogen activator inhibitor‑1 expression in a mouse model of cerebral infarction. These results suggested that aspirin may improve cerebral infarction by downregulating TLR4/NF‑κB‑mediated ER stress in a mouse model.

Cyanidin-3-O-glucoside promotes the biosynthesis of progesterone through the protection of mitochondrial function in Pb-exposed rat leydig cells

Cyanidin-3-O-glucoside (C3G) is an anthocyanin that has been reported to reduce the toxicity of heavy metals. In the present study, the protection effects of C3G on the biosynthesis of progesterone, the precursor of testosterone, against lead (Pb) in R2C rat Leydig cells were examined. Treatment of R2C cells with 100 μM Pb resulted in a significant decrease in progesterone production. After being cultured in a medium containing C3G and Pb, R2C cells exhibited an increase in progesterone concentration compared with the Pb treatment, as a result of up-regulation of the expression of the steroidogenic enzymes steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 enzyme cholesterol side chain cleavage enzyme (CYP11A1). Pb-induced inhibition of extracellular regulated Kinase 1/2 (ERK 1/2) phosphorylation and down-regulation of protein kinase A (PKA) expression were alleviated by C3G. In addition, Pb-induced reactive oxygen species (ROS) overproduction led to mitochondrial depolarization, resulting in a decrease in progesterone biosynthesis, while C3G intervention reduced the ROS level and increased progesterone production. In conclusion, C3G may alleviate the Pb-induced decrease of progesterone biosynthesis by modulating the dysfunction of mitochondria, including decreasing oxidative stress and regulating expression of steroidogenic enzyme proteins.

Hyperexpression of Integrin α5β1 Promotes Resistance of MCF-7 Human Breast Carcinoma Cells to Doxorubicin via ERK Protein Kinase Down-regulation.

In MCF-7 human breast carcinoma cells, α5β1 integrin hyperexpression, which was accomplished by transduction of a full-length α5 integrin cDNA, increased by about 50-70% the number of cells, survived during 48-72 h cell treatment with doxorubicin. Up-regulation of α5β1 reduced the level of the apoptogenic p53 protein and p21 cell cycle inhibitor, but enhanced the activity of Akt and mTOR protein kinases. In addition to these findings, we observed a significant decrease in the activity of both isoforms of phosphokinase Erk1/2, which is known to play a key role in cell viability pathways, including pathways alleviating stress damages caused by distinct antitumor drugs. Diminished Erk activity accompanying the rise of drug resistance can be explained by an "atypical" function of this kinase, which, in the cells studied, promotes an enhanced rather than reduced sensitivity to doxorubicin. To verify this suggestion, the effect of a specific Erk inhibitor, PD98059, on the resistance to doxorubicin of control and α5 cDNA-transduced MCF-7 cells was investigated. The data showed that suppression of Erk activity increased the resistance of control cells (transduced with an "empty" vector) to a level higher than that demonstrated by the α5 cDNA-transduced cells. The highest level of resistance was observed in α5β1-trancduced cells treated with PD98059. Akt and mTOR kinase inhibitors had little if any effect on doxorubicin resistance of α5 cDNA-transduced MCF-7 cells. The data show for the first time that integrin α5β1 can stimulate drug resistance of tumor cells through a mechanism based on the inhibition of protein kinase Erk. From a more general view, the results of this investigation suggest that signal protein kinases can perform in tumor cells "non-canonical" functions, opposite to those, which are the basis for using kinase inhibitors in targeted cancer therapy. It follows that if a protein kinase is supposed to be used as a target for such therapy, it is important to explore its features in the particular tumor prior to the onset of treatment.