Adenosine Monophosphate Response Element-binding Protein Research Articles

Effects of Grape Seed Extract and Proanthocyanidin B2 on In Vitro Proliferation, Viability, Steroidogenesis, Oxidative Stress, and Cell Signaling in Human Granulosa Cells.

Reactive oxygen species (ROS) which lead to oxidative stress affect ovarian function. Grape seed extract (GSE) could be proposed as an effective antioxidant, particularly due to its proanthocyanidin content. In this study, we investigated a dose effect (0, 0.01, 0.1, 1, 10, 50, and 100 μg/mL) of GSE and proanthocyanidin B2 (GSPB2) on the ROS content, cell proliferation, cell viability, and steroidogenesis in both primary luteinized granulosa cells (hGC) and the tumor granulosa cell line (KGN). The levels of ROS were measured using ROS-Glo assay. Cell proliferation and viability were evaluated by [3H]-thymidine incorporation and Cell Counting Kit-8 (CCK8) assay, respectively. Steroid secretion was evaluated by radioimmunoassay. We also analyzed the cell cycle component protein level and signaling pathways by immunoblot and the NOX4 mRNA expression by RTqPCR. From 0.1 to 1 μg/mL, GSE and GSBP2 reduced the ROS cell content and the NOX4 mRNA levels, whereas, GSE and GSBP2 increased the ROS cell content from 50 to 100 μM in both hGC and KGN. GSE and GSPB2 treatments at 50 and 100 μg/mL induced a delay in G1 to S phase cell cycle progression as determined by fluorescence-activated cell sorting. Consequently, they reduced cell growth, cyclin D2 amount, and Akt phosphorylation, and they increased protein levels of p21 and p27 cyclin-dependent kinase inhibitors. These data were also associated with an increase in cell death that could be due to a reduction in Bcl-2-associated death promoter (BAD) phosphorylation and an increase in the cleaved-caspase-3 level. All these negative effects were not observed at lower concentrations of GSE and GSPB2 (0.01 to 10 μg/mL). Interestingly, we found that GSE and GSPB2 treatments (0.1 to 100 μg/mL) improved progesterone and estradiol secretion and this was associated with a higher level of the cholesterol carriers, StAR (steroidogenic acute regulatory protein), CREB (Cyclic adenosine monophosphate Response Element-binding protein), and MAPK ERK1/2 (Mitogen-Activated Protein Kinases Extracellular signal-Regulated Kinases 1/2) phosphorylation in both hGC and KGN cells. Taken together, GSE and GSPB2 (0.1–10 μg/mL) in vitro treatments decrease oxidative stress and increase steroidogenesis without affecting cell proliferation and viability in human granulosa cells.

Long-term Surgical and Chemical Castration Deteriorates Memory Function Through Downregulation of PKA/CREB/BDNF and c-Raf/MEK/ERK Pathways in Hippocampus.

PurposeGoserelin is a drug used for chemical castration. In a rat model, we investigated whether surgical and chemical castration affected memory ability through the protein kinase A (PKA)/cyclic adenosine monophosphate response element-binding protein (CREB)/brain-derived neurotrophic factor (BDNF) and c-Raf/mitogen-activated protein kinases-extracellular signal–regulated kinases (MEK)/extracellular signal–regulated kinases (ERK) pathways in the hippocampus.MethodsOrchiectomy was performed for surgical castration and goserelin acetate was subcutaneously transplanted into the anterior abdominal wall for chemical castration. Immunohistochemistry was done to quantify neurogenesis. To assess the involvement of the PKA/CREB/BDNF and c-Raf/MEK/ERK pathways in the memory process, western blots were used.ResultsThe orchiectomy group and the goserelin group showed less neurogenesis and impaired short-term and spatial memory. Phosphorylation of PKA/CREB/BDNF and phosphorylation of c-Raf/MEK/ERK decreased in the orchiectomy and goserelin groups.ConclusionsShort-term memory and spatial memory were affected by surgical and chemical castration via the PKA/CREB/BDNF and c-Raf/MEK/ERK signaling pathways.

Adipokine zinc-α2-glycoprotein alleviates lipopolysaccharide-induced inflammatory responses through the β3-AR/PKA/CREB pathway

Humans and animals frequently experience dysmetabolism induced by inflammation. Zinc-α2-glycoprotein (ZAG), a newly identified adipokine, is potentially involved in lipid metabolism. Our previous study revealed that the ZAG content increased after lipopolysaccharide (LPS) treatment. To clarify ZAG’s possible effects on inflammatory responses and lipid metabolism, we used gene overexpression and knockout mice as models to investigate the function of ZAG during inflammation. The results showed that LPS increased plasma triglyceride, non-esterified fatty acid and hepatic triglyceride, while ZAG overexpression decreased these effects. Furthermore, ZAG overexpression weakened inflammatory responses, suppressed lipogenesis, and improved mitochondrial function during inflammation. ZAG overexpression also increased β3-adrenoreceptor, protein kinase A, and phosphorylated cyclic adenosine monophosphate-response element binding protein (CREB), promoted the combination of CREB and CREB-binding protein (CBP), and competitively inhibited the combination of nuclear factor-κB and CBP. After ZAG knockout, LPS-induced the hyperlipidemia worsened. ZAG knockout aggravated inflammatory responses, promoted lipogenesis, and weakened mitochondrial function during inflammation. ZAG knockout also decreased β3-adrenoreceptor and protein kinase A. The present study demonstrated that ZAG alleviated lipid metabolism disorders by weakening inflammatory responses. The β3-adrenoreceptor/protein kinase A/CREB pathway mediated the effects of ZAG on inflammation. These results will provide new insight for research on anti-inflammation.

Combination of treadmill exercise with bone marrow stromal cells transplantation activates protein synthesis-related molecules in soleus muscle of the spinal cord injured rats.

The present study investigated whether treadmill exercise with bone marrow stromal cells (BMSCs) transplantation increase expression level of protein synthesis-related molecules in the soleus muscle after spinal cord injury (SCI). The spinal cord contusion injury was performed at the T9–10 level using the impactor (10 g×25 mm). BMSCs were cultured from femur and tibia of 4-week-old rats and then transplanted directly into the lesion 1-week post injury. The rats in exercise group were walking on treadmill device for 6 days per a week during 6 weeks. Prepared soleus muscles were used for examining mechanisms of protein synthesis after SCI. Myostatin induction level was increased by SCI, but BMSCs engrafting after SCI decreased compared to SCI group. Combination of treadmill exercise with BMSCs showed more potent decrement on myostatin expression. Protein kinase B (Akt) and mammalian target of rapamycin (mTOR) levels were significantly increased in SCI and BMSCs transplantation group compared to SCI group. Combination of treadmill exercise with BMSCs further facilitated expression levels of Akt and mTOR. Insulin-like growth factor-I (IGF-I) and phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB) induction levels were more increased in SCI and BMSC transplantation group compared to SCI group. Combination of treadmill exercise with BMSCs further increased expression levels of IGF-I and p-CREB, although statistical significance was not appeared. Combining treadmill exercise with BMSCs transplantation might accelerate protein synthesis and hypertrophy in the soleus muscle after SCI through activation of IGF-I/mTOR signaling pathway.

Additive antidepressant-like effects of fasting with β-estradiol in mice.

Our recent study has shown that acute fasting produces antidepressant‐like effects in male mice. However, there is little evidence regarding the antidepressant‐like effects of acute fasting in female mice. Moreover, it is not yet clear whether estrogen produces additive effects with acute fasting. Therefore, this study aims to investigate the antidepressant‐like effects of acute fasting plus estrogen treatment. In this study, the acute fasting produced antidepressant‐like effects in female mice and the antidepressant‐like effects of 9 hours fasting with those of β‐estradiol (E2) were additive. Activity of the cyclic adenosine monophosphate (cAMP) response element‐binding protein (CREB)‐brain‐derived neurotrophic factor (BDNF) pathway in the prefrontal cortex (PFC) and hippocampus (HP) was increased, as well as neurogenesis in the subgranular zone of the hippocampus. Serum ghrelin and estrogen were also increased by fasting plus E2. Furthermore, RNA‐seq analysis indicated that fasting and E2 co‐regulate similar gene expression pathways, underlying similar neurological functions. Taken together, these data suggest that E2 produces additive antidepressant‐like effects with fasting by activating the CREB‐BDNF pathway in the PFC and HP. Genome‐wide transcriptome mapping suggests that fasting may be used as an adjunct to estrogen replacement therapy for the treatment of depression associated with reduced estrogen function.

The GLP-1 agonist, liraglutide, ameliorates inflammation through the activation of the PKA/CREB pathway in a rat model of knee osteoarthritis

BackgroundInflammation is a common pathological phenomenon of osteoarthritis (OA). Accumulated evidence indicates that ameliorating or suppressing inflammation might be a promising and effective therapeutic strategy for the treatment of OA. Notably, glucagon-like peptide-1 (GLP-1)-based drugs are being successfully used to control glucose levels in patients with diabetes mellitus. In addition, recent findings have indicated that GLP-1 agonists, such as liraglutide have therapeutic potential in preventing inflammation-related disorders through the regulation of protein kinase A (PKA)/ cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) signals. Intra-articular injection of monoiodoacetate (MIA) has been widely used to induce OA. Thus, the present study aimed to investigate whether liraglutide has anti-inflammatory effects on MIA-induced OA rats and uncover its underlying molecular mechanisms.MethodsIntra-articular injection of MIA was used to induce knee OA in a rat model. Subcutaneous injection of liraglutide was used to upregulate the expression of GLP-1 receptor (GLP-1R). Western blot analysis was utilized to measure the expression of GLP-1R, PKA/CREB pathway components and inflammation-related proteins, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6. Immunoprecipitation techniques were used to detect the interactions between GLP-1R and the PKA/CREB pathway.ResultsThe levels of GLP-1R decreased significantly in the knees of OA rats, accompanied by the downregulation of PKA /CREB signals and upregulation of inflammation-related proteins. We also found that GLP-1R interacted with the PKA/CREB pathway and that liraglutide could activate PKA/CREB signals, thereby inhibiting the expression of inflammation-related proteins.ConclusionsTogether our results suggest that liraglutide exhibits anti-inflammatory activity through the activation of the PKA/CREB pathway in an OA rat model.

Relationship between normal weight obesity and mild cognitive impairment is reflected in cognitive-related genes in human peripheral blood mononuclear cells.

Obesity contributes to the development of mild cognitive impairment, but the potential role of normal weight obesity in this disease has not been explored in humans. The aim of the study was to reveal the relationship between normal weight obesity and mild cognitive impairment in elderly individuals. This study consisted of 360 patients with amnestic mild cognitive impairment and 360 cognitively normal controls. Normal weight obesity was defined as having metabolic syndrome but a normal weight. Metabolic health meant having no metabolic syndrome. Reverse transcription quantitative real-time polymerase chain reaction was adopted to measure the messenger RNA expression of four cognitive-related genes (amyloid precursor protein, cyclic adenosine monophosphate-responsive element-binding protein 1, sortilin-related receptor 1, and synapsin I) in peripheral blood mononuclear cells. Normal weight obesity was related to a higher risk of amnestic mild cognitive impairment (odds ratio = 3.14, 95% confidence interval: 2.13-4.60). In the patients, the expression of each gene in the peripheral blood mononuclear cells was linearly related to Mini-Mental State Examination and Montreal Cognitive Assessment scores (P < 0.05). The expression of these genes in the patients with metabolic health deviated from the normal levels found in the controls (P < 0.05), and the deviations were more significant in the patients with normal weight obesity (P < 0.05). Normal weight obesity may be a potential risk factor for amnestic mild cognitive impairment in elderly. This relationship was reflected in the abnormal expression of several cognitive-related genes in peripheral blood mononuclear cells.

PKA- and Ca2+-dependent p38 MAPK/CREB activation protects against manganese-mediated neuronal apoptosis

Although manganese (Mn) is an essential trace element, its excessive consumption may lead to neuronal death and neurodegenerative disorders. Human cells launch adaptive responses to attenuate Mn-induced neurotoxicity. However, the regulation of the responsive proteins and their function during Mn-stimulated neurotoxicity remain largely unknown. We report the role of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in Mn-induced neuronal apoptosis. Mn increased CREB phosphorylation and cellular apoptosis in both PC12 cells and mouse brain tissue. Furthermore, downregulation of CREB with shRNA plasmid transfection significantly worsened the PC12 cell apoptosis by decreasing mRNA and protein expression of brain-derived neurotrophic factor (BDNF). Moreover, Mn enhanced protein kinase A (PKA) activation and activation of the p38 MAPK and JNK pathways. Inhibition of p38 MAPK rather than JNK effectively reduced the CREB phosphorylation. Subsequent analysis showed that a PKA inhibitor blocked p38 MAPK and CREB phosphorylation. Moreover, the intracellular Ca2+ chelator BAPTA-AM decreased the phosphorylation of p38 MAPK and CREB but failed to reduce PKA activation. In summary, p38 MAPK/CREB activation via PKA activation and increased cellular Ca2+ helped to alleviate Mn-induced neuronal apoptosis via BDNF regulation. These findings improve our understanding of Mn-induced neurotoxicity and the molecular targets to antagonise it.

ILF2 Directly Binds and Stabilizes CREB to Stimulate Malignant Phenotypes of Liver Cancer Cells.

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is overexpressed and has an oncogenic role in hepatocellular carcinoma (HCC). Interleukin enhancer binding factor 2 (ILF2) has become research hotspot in liver cancer recently. However, it is still unclear whether and how CREB and ILF2 interact with each other. And how this interaction exerts its role in occurrence and development of liver cancer is still unclear. Here, we found that ILF2 directly bound with CREB, and this binding was essential for the malignant phenotypes of liver cancer cells. Moreover, we found that ILF2 acted as one of the upstream proteins of CREB and promoted CREB only in the protein level, whereas ILF2 expression was not regulated by CREB. Mechanistically, ILF2 bound to the pKID domain of CREB and stimulated its phosphorylation at Ser133. Taken together, our study finds a novel interaction between CREB and ILF2 in liver cancer, and this interaction might play a role in the diagnosis and remedy of liver cancer.

Optimized liposomes with transactivator of transcription peptide and anti-apoptotic drugs to target hippocampal neurons and prevent tau-hyperphosphorylated neurodegeneration

Liposomes (lip) carrying pharmaceuticals have shown promise in their ability to advance the therapy for neurodegenerative diseases. However, the low nerve-targeting capacity and poor penetration rate of lip through the blood–brain barrier (BBB) are major hurdles to achieving successful treatment. Herein, we developed lip incorporating cardiolipin (CL) and phosphatidic acid (PA) to promote their capability against hyperphosphorylation of tau protein, and a transactivator of transcription (TAT) peptide to permeate the BBB for delivering nerve growth factor (NGF), rosmarinic acid (RA), curcumin (CURC) and quercetin (QU). We derived an optimization method to assess a better composition of phospholipids in the lip loaded with the four medicines. Experimental results revealed that this optimized lip increased the viability of SK-N-MC cells insulted with β-amyloid peptide (Aβ) fibrils and prevented Wistar rat brain from producing hyperphosphorylated tau. CL and PA and the grafted TAT peptide on the carrier surface improved the rescue efficiency by inhibiting Aβ deposition and reducing the expressions of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), c-Jun N-terminal protein kinase, p38, tau at serine 202 and caspase-3. The lip also enhanced the expressions of p-ERK5 and p-cyclic adenosine monophosphate response element-binding protein. The amalgamated activity of NGF, RA, CURC and QU, and the effect of charged CL/PA on Aβ deposits supported the therapeutic efficacy of lip. The optimized TAT-NGF-RA-CURC-QU-CL/PA-lip can be a capable drug delivery system to cross the BBB and protect Alzheimer’s disease brains from tau hyperphosphorylation. Statements of SignificanceThe therapeutic efficiency of liposomes (lip) against neurodegenerative disorder depends on their nerve-targeting capacity and ability to permeate the blood–brain barrier (BBB). Lip was developed incorporating cardiolipin (CL) and phosphatidic acid (PA) to promote their target specificity against hyperphosphorylation of tau protein, and a transactivator of transcription (TAT) peptide to permeate the BBB. We have successfully derived an optimization method using a new mathematical expression for the first time to assess a better composition of phospholipids in lip loaded with nerve growth factor (NGF), rosmarinic acid (RA), curcumin (CURC) and quercetin (QU). The optimized TAT-NGF-RA-CURC-QU-CL/PA-lip efficaciously down-regulated the expressions of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), c-Jun N-terminal protein kinase, p38, tau at serine 202 and caspase-3, and up-regulated the expressions of p-ERK5 and p-cyclic adenosine monophosphate response element-binding protein in Alzheimer’s disease Wistar rat model.

Blockade of NMDA Receptors and Nitric Oxide Synthesis Potentiated Morphine-Induced Anti-Allodynia via Attenuating Pain-Related Amygdala pCREB/CREB Signaling Pathway

The present study investigated the role of the amygdala N-methyl-d-aspartate (NMDA) receptors/nitric oxide synthase pathway in morphine-induced anti-allodynia. Concurrently with the bilateral cannulation of the central amygdala, chronic constriction of the sciatic nerve was performed on male Wistar rats. Morphine (3–5 mg/kg) was administered intraperitoneally to induce anti-allodynia. When D-AP5, a selective NMDA receptor antagonist, (.05–.1 µg/rat) or NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME), the nitric oxide synthase inhibitor (.1–.5 µg/rat), were microinjected into the central amygdala, the higher doses potentiated an ineffective dose of morphine (3 mg/kg). Microinjection of the same doses of D-AP5 and L-NAME without morphine had no effect. Comicroinjection of the ineffective doses of L-NAME (.1 µg/rat) and D-AP5 (.05 µg/rat) with a 5-minute interval, enhanced the anti-allodynic effect of morphine (3 mg/kg). Western blot analysis was employed to evaluate the levels of cyclic adenosine monophosphate-response element-binding protein (CREB) and phosphorylated CREB (pCREB) in the amygdala tissues. Our results showed that neuropathic pain increased the pCREB/CREB ratio in the amygdala, and this ratio was decreased after morphine-induced anti-allodynia. The potentiative effect of the coadministration of D-AP5/L-NAME on an ineffective dose of morphine also decreased the amygdala pCREB/CREB levels. Therefore, it seems that the amygdala pCREB/CREB signaling pathway plays a critical role in processing neuropathic pain. Moreover, the glutamate NMDA receptors and nitric oxide system in the amygdala may be involved in morphine-induced anti-allodynia. PerspectiveNeuropathic pain is difficult to treat and the exact mechanisms remain unknown. This article suggests the importance of the amygdala glutamatergic and nitric oxide systems in morphine-induced anti-allodynia. These findings might be used in clinical studies to reach a better understanding of neuropathic pain mechanisms and treatment.

Novel protective effect of O-1602 and abnormal cannabidiol, GPR55 agonists, on ER stress-induced apoptosis in pancreatic β-cells

Insulin resistance and β-cell dysfunction are the main defects in Type 2 Diabetes Mellitus (T2DM), and β-cell dysfunction and apoptosis is the critical determinant in the progression of T2DM. G-protein coupled receptor 55 (GPR55) is an orphan G-protein coupled receptor, which is activated by endocannabinoids and lipid transmitters. Recently, GPR55 was shown to regulate glucose and energy homeostasis, however its role in β-cell apoptosis was not studied. Therefore, in this study, we investigated the novel effect of GPR55 agonists, O-1602 and abnormal cannabidiol (Abn-CBD), on endoplasmic reticulum (ER) stress-induced apoptosis in mouse pancreatic β-cell lines, MIN6 and Beta-TC-6, and its underlying mechanisms. Our results showed that O-1602 and Abn-CBD reduced ER stress-induced apoptosis in MIN6 and Beta-TC-6 cells. This was through the phosphorylation of 3′-5′-cyclic adenosine monophosphate response element-binding protein (CREB) in β-cells, hence activating CREB downstream anti-apoptotic genes, Bcl-2 and Bcl-xL. Moreover, O-1602 and Abn-CBD directly activated kinases, CaMKIV, Erk1/2 and PKA, to induce CREB phosphorylation. Therefore, our results indicated that GPR55 agonists protected from β-cell apoptosis through CREB activation, thus up-regulating anti-apoptotic genes. In conclusion, our study provided a novel protective effect of GPR55 agonists on ER stress-induced apoptosis in β-cells and its underlying mechanisms mediating this protection, therefore we suggested that GPR55 might be a therapeutic target for T2DM.

Chemical Constituents and Antidepressant-Like Effects in Ovariectomized Mice of the Ethanol Extract of Alternanthera philoxeroides.

The previously unreported flavone glycoside, demethyltorosaflavone B (2) and the E-propenoic acid substituted flavone, torosaflavone E (3a), were isolated together with nine previously reported metabolites, including indole-3-carbaldehyde, oleanonic acid, vanillic acid, p-hydroxybenzoic acid, altheranthin (1a), alternanthin B (1b), demethyltorosaflavone D (3b), luteolin 8-C-E-propenoic acid (4) and chrysoeriol 7-O-rhamnoside (5), from the ethanol extract of the aerial part of Althernanthera philoxeroides. The crude ethanol extract was evaluated for its in vitro estrogenic activity in MCF-7 breast cancer cell line. The crude ethanol extract was also investigated in vivo for its antidepressant-like effects on ovariectomized mice using tail suspension and forced swimming tests, while its effect on the locomotor activity was evaluated by a Y-maze test. The effect of the crude extract on the serum corticosterone level, size and volume of uterus of the ovariectomized mice were also investigated. The expression of the mouse cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF) and β-actin mRNAs in hippocampus and frontal cortex was also evaluated, using semiquantitative reverse transcription-polymerase chain reaction. The crude extract and the isolated compounds 1a, 1b, 3a, 3b and 5, were evaluated for their inhibitory effects on monoamine oxidases (MAOs)-A and -B.

Thyroxin Protects White Matter from Hypoxic-Ischemic Insult in the Immature Sprague⁻Dawley Rat Brain by Regulating Periventricular White Matter and Cortex BDNF and CREB Pathways.

Background: Periventricular white-matter (WM) injury is a prominent feature of brain injury in preterm infants. Thyroxin (T4) treatment reduces the severity of hypoxic-ischemic (HI)-mediated WM injury in the immature brain. This study aimed to delineate molecular events underlying T4 protection following periventricular WM injury in HI rats. Methods: Right common-carotid-artery ligation, followed by hypoxia, was performed on seven-day-old rat pups. The HI pups were injected with saline, or 0.2 or 1 mg/kg of T4 at 48–96 h postoperatively. Cortex and periventricular WM were dissected for real-time (RT)-quantitative polymerase chain reactions (PCRs), immunoblotting, and for immunofluorescence analysis of neurotrophins, myelin, oligodendrocyte precursors, and neointimal. Results: T4 significantly mitigated hypomyelination and oligodendrocyte death in HI pups, whereas angiogenesis of periventricular WM, observed using antiendothelium cell antibody (RECA-1) immunofluorescence and vascular endothelium growth factor (VEGF) immunoblotting, was not affected. T4 also increased the brain-derived neurotrophic factors (BDNFs), but not the nerve growth factor (NGF) expression of injured periventricular WM. However, phosphorylated extracellular signal regulated kinase (p-ERK) and phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB) concentrations, but not the BDNF downstream pathway kinases, p38, c-Jun amino-terminal kinase (c-JNK), or Akt, were reduced in periventricular WM with T4 treatment. Notably, T4 administration significantly increased BDNF and phosphorylated CREB in the overlying cortex of the HI-induced injured cortex. Conclusion: Our findings reveal that T4 reversed BNDF signaling to attenuate HI-induced WM injury by activating ERK and CREB pathways in the cortex, but not directly in periventricular WM. This study offers molecular insight into the neuroprotective actions of T4 in HI-mediated WM injury in the immature brain.

Effects of muscarinic acetylcholine receptor stimulation on the differentiation of mouse induced pluripotent stem cells into neural progenitor cells.

Muscarinic acetylcholine receptors (mAchRs), which are expressed in various embryonic cells, may regulate neuronal differentiation. In the present study, we examined the effects of mAchR stimulation on the differentiation of mouse induced pluripotent stem (iPS) cells into neural progenitor cells (NPCs). Mouse iPS cells were cultured on ultra-low attachment dishes to induce embryoid body (EB) formation. All-trans retinoic acid (ATRA, 3μmol/L) and/or pilocarpine (10 or 100μmol/L), a mAchR agonist, were added to EB cultures for 4days, following which the EBs were cultured on gelatin-coated plates for 7days. Subtype-specific antibody staining revealed that mouse iPS cells predominantly express m2 - and m4 -AchR. Treatment with pilocarpine alone did not affect the expression of Nestin (a specific marker for neural progenitor cells). However, additional treatment with pilocarpine significantly suppressed ATRA-induced Nestin expression. Pretreating EBs with either AF-DX116 (an antagonist of both m2 - and m4 -AchR) or forskolin (an activator of adenylate cyclase) significantly reversed the pilocarpine-induced suppression of Nestin expression. In addition, treatment with pilocarpine significantly suppressed ATRA-induced phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). These findings suggest that the stimulation of m2 - or m4 -AchR suppresses ATRA-induced differentiation of mouse iPS cells into NPCs by inhibiting the cAMP/protein kinase A pathway and CREB activation.

Misaligned Feeding May Aggravate Pain by Disruption of Sleep-Awake Rhythm.

Increasing evidence suggests that patients with eating disorders are more likely to develop chronic pain. A misaligned diet has been reported to disrupt the sleep-awake rhythms. Combined with our previous investigation on circadian pain, we aimed to investigate the role of misaligned diet in the pain sensitivity and the underlying mechanisms. Two-month-old C57BL/6J male mice were administered chronic constriction injury (CCI) surgery to establish neuropathic pain models. CCI mice were randomized to scheduled food access throughout the whole day (CCI-free), during the daytime (CCI-misaligned), and at night (CCI-aligned), respectively. The paw withdrawal mechanical threshold, indicating pain behavior, was measured by Von Frey. The gross motor activity pattern indicating the sleep-awake rhythm was monitored by Mini-Mitter. Melatonin (Mel) was administered to ameliorate the sleep-awake rhythm (CCI-free + Mel and CCI-misaligned + Mel). The expressions of circadian pain-related proteins were detected by quantitative polymerase chain reaction and western blot. The primary outcome is the pain threshold and the secondary outcome is the sleep-awake rhythm. Misaligned diet during the peri-CCI surgery period significantly decreased the paw withdrawal mechanical threshold compared with the CCI-free mice (day 14: 0.40 ± 0.09 vs 0.64 ± 0.15; P = .03;) and altered the sleep-awake rhythm. Mel pretreatment alleviated the increased pain (day 14, CCI-misaligned + Mel versus CCI-misaligned: day 14: 0.60 ± 0.13 vs 0.35 ± 0.12; P = .022) and the disrupted sleep-awake rhythm caused by misaligned feeding. The mRNA levels of N-methyl-D-aspartate receptor subtype 2B (NR2B), Ca/calmodulin-dependent protein kinase II (CaMKII), and cyclic adenosine monophosphate-response element binding protein (CREB) in the spinal dorsal horn increased in CCI-misaligned mice compared with the CCI-free mice. The phosphor-NR2B, phosphor-CaMKII, and phosphor-CREB also increased in CCI-misaligned mice compared with the CCI-free mice. However, the expressions of NR2B, CaMKII, and CREB were decreased in CCI-misaligned + Mel mice compared to CCI-misaligned mice at both transcriptional and translational levels. Misaligned diet might aggravate pain sensitivity through the disruption of the sleep-awake cycle, which could be recovered by Mel. NR2B-CaMKII-CREB may participate in the disruption of sleep-awake rhythm-mediated pain aggravation.

Order-disorder transition of intrinsically disordered kinase inducible transactivation domain of CREB.

Transcription factor cyclic Adenosine monophosphate response-element binding protein plays a critical role in the cyclic AMP response pathway via its intrinsically disordered kinase inducible transactivation domain (KID). KID is one of the most studied intrinsically disordered proteins (IDPs), although most previous studies focus on characterizing its disordered state structures. An interesting question that remains to be answered is how the order-disorder transition occurs at experimental conditions. Thanks to the newly developed IDP-specific force field ff14IDPSFF, the quality of conformer sampling for IDPs has been dramatically improved. In this study, molecular dynamics (MD) simulations were used to study the order-to-disorder transition kinetics of KID based on the good agreement with the experiment on its disordered-state properties. Specifically, we tested four force fields, ff99SBildn, ff99IDPs, ff14IDPSFF, and ff14IDPs in the simulations of KID and found that ff14IDPSFF can generate more diversified disordered conformers and also reproduce more accurate experimental secondary chemical shifts. Kinetics analysis of MD simulations demonstrates that the order-disorder transition of KID obeys the first-order kinetics, and the transition nucleus is I127/L128/L141. The possible transition pathways from the nucleus to the last folded residues were identified as I127-R125-L138-L141-S143-A145 and L128-R125-L138-L141-S143-A145 based on a residue-level dynamical network analysis. These computational studies not only provide testable prediction/hypothesis on the order-disorder transition of KID but also confirm that the ff14IDPSFF force field can be used to explore the correlation between the structure and function of IDPs.

Role of melatonin receptor 1A and pituitary homeobox-1 coexpression in protecting tubular epithelial cells in membranous nephropathy.

Membranous nephropathy (MN), a type of glomerular nephritis, is one of the most common causes of nephrotic syndrome in adults. Although it is known that melatonin plays a protective role in MN, the role of melatonin receptors in the pathophysiology of MN is unclear. Using an experimental MN model and clinical MN specimens, we studied melatonin receptor expression and found that melatonin receptor 1A (MTNR1A) expression was significantly downregulated in renal tubular epithelial cells. Molecular studies showed that the transcription factor pituitary homeobox-1 (PITX1) promoted MTNR1A expression via direct binding to its promoter. Treatment of a human tubular cell line with albumin to induce injury resulted in the stable reduction in MTNR1A and PITX1 expression. PITX1 levels were significantly downregulated in tubular epithelial cells from mice MN kidneys and MN renal specimens. Knockdown of MTNR1A, PITX1, or cyclic adenosine monophosphate-responsive element-binding protein (CREB) decreased E-cadherin (CDH1) expression, but upregulated Per2 and α-smooth muscle actin (αSMA) expression. Blockade of the MTNR1A receptor with luzindole in MN mice further impaired renal function; this was accompanied by CDH1 downregulation and Per2 and αSMA upregulation. Together, our results suggest that in injured tissue, decreased PITX1 expression at the MTNR1A promoter regions leads to decreased levels of MTNR1A in renal tubular epithelial cells, which increases the future risk of MN.

The Melatonin Signaling Pathway in a Long-Term Memory In Vitro Study.

The activation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) via phosphorylation in the hippocampus is an important signaling mechanism for enhancing memory processing. Although melatonin is known to increase CREB expression in various animal models, the signaling mechanism between melatonin and CREB has been unknown in vitro. Thus, we confirmed the signaling pathway between the melatonin receptor 1 (MT1) and CREB using melatonin in HT-22 cells. Melatonin increased MT1 and gradually induced signals associated with long-term memory processing through phosphorylation of Raf, ERK, p90RSK, CREB, and BDNF expression. We also confirmed that the calcium, JNK, and AKT pathways were not involved in this signaling pathway by melatonin in HT-22 cells. Furthermore, we investigated whether melatonin regulated the expressions of CREB-BDNF associated with long-term memory processing in aged HT-22 cells. In conclusion, melatonin mediated the MT1-ERK-p90RSK-CREB-BDNF signaling pathway in the in vitro long-term memory processing model and increased the levels of p-CREB and BDNF expression in melatonin-treated cells compared to untreated HT-22 cells in the cellular aged state. Therefore, this paper suggests that melatonin induces CREB signaling pathways associated with long-term memory processing in vitro.

Stimulation of ACE2/ANG(1-7)/Mas Axis by Diminazene Ameliorates Alzheimer's Disease in the D-Galactose-Ovariectomized Rat Model: Role of PI3K/Akt Pathway.

Overactivation of angiotensin-converting enzyme/angiotensin 2/angiotensin receptor-1 (ACE/Ang2/AT1) axis provokes amyloid-β-induced apoptosis and neurodegeneration in Alzheimer's disease (AD). Moreover, activation of AT1 impairs the survival pathway phosphoinositide 3-kinase/protein kinase B (PI3K/Akt). Interestingly, the coupling between ACE2/Ang(1-7)/Mas receptor (MasR) axis and PI3K/Akt activation opposes AT1-induced apoptosis. However, the effect of in vivo stimulation of MasR against AD and its correlation to PI3K/Akt is not yet elucidated. Thus, the present study aimed to investigate the relationship between PI3K/Akt pathway and the activation of ACE2/MasR in the AD model of D-galactose-ovariectomized rats. AD features were induced following 8-week injection of D-galactose (150mg/kg, i.p.) in ovariectomized female rats. The ACE2 activator dimenazine (15mg/kg, i.p.) was daily administered for 2months. DIZE administration boosted the hippocampal expression of ACE2 and Mas receptors while suppressing AT1 receptor. Notably, dimenazine enhanced the expression of phosphorylated survival factors (PI3K, Akt, signal transducer, and activator of transcription-3) and neuroplasticity proteins such as cyclic adenosine monophosphate-responsive element-binding protein and brain-derived neurotrophic factor along with nicotinic and glutamatergic receptors. Such effects were accompanied by suppressing phosphorylated tau and glycogen synthase kinase3β along with caspase-3, cytochrome-c, nuclear factor kappa B, tumor necrosis factor alpha, and glial fibrillary acidic protein contents. Dimenazine ameliorated the histopathological damage observed in D-galactose-ovariectomized rats and improved their learning and recognition memory in Morris water maze and novel object recognition tests. In conclusion, dimenazine-induced stimulation of ACE2/Ang(1-7)/Mas axis subdues cognitive deficits in AD most probably through activation of PI3K/Akt pathway.