Response Element-binding Protein Research Articles

Long-term treatment with roflumilast improves learning of fear extinction memory and anxiety-like response in a type-1 diabetes mellitus animal model

Diabetic encephalopathy is related to serious damage to the Central Nervous System leading to several disturbances in memory processing and emotions. It is known that the cyclic adenosine 3′,5′-monophosphate (cAMP) responsive element-binding protein (CREB) pathway participates in neuronal plasticity and prevention of neuroinflammation, as well as the mediation of learning/memory processes and emotions in brain areas such as the hippocampus (HIP) and prefrontal cortex (PFC). We aimed to investigate the effect of acute (one injection) and long-term treatment (21 days) with roflumilast (ROF; i.p.; 0, 0.01, 0.03, 0.1 mg/kg), a drug able to inhibit the enzyme phosphodiesterase-4 (PDE-4) responsible for cAMP hydrolysis, on parameters related to the acquisition of fear extinction memory and anxiety-like responses in animals with type-1 diabetes mellitus (T1DM) induced through one injection of streptozotocin (60 mg/kg; ip; STZ animals). When we performed acute treatment, no difference was observed between all the groups when resubmitted to the same context paired with an aversive stimulus (footshock) or to a neutral context. In contrast, long-term treatment was able to improve learning of extinction fear memory and discriminating between a conditioned and neutral context. Moreover, this treatment decreased the pronounced anxiety-like response of STZ animals. In addition, there was an increase in the product of the CREB signaling pathway, the pro brain-derived neurotrophic factor, in the HIP and PFC of these animals. The treatment did not impair glycemic control, whereas it decreased the animal’s blood glucose levels. To conclude, these findings suggest that ROF treatment repositioning has potential for future translational investigations involving diabetic patients considering its beneficial effects on emotional processes related to fear memory and anxiety, in addition to improvement of glycemic control.

Yuk-Gunja-Tang attenuates neuronal death and memory impairment via ERK/CREB/BDNF signaling in the hippocampi of experimental Alzheimer's disease model.

Yuk-Gunja-Tang (YG) is the Korean traditional medicine in East Asia for gastrointestinal disorders. In the present study, we determined the protective effects of YG on glutamate-induced cytotoxicity in HT22 hippocampal neuronal cells and mice with scopolamine-induced memory impairment. In vitro assessments were performed using a cell viability assay, flow cytometry, and Western blotting, while in vivo assessments were performed in C57BL/6 mice administered with YG for 7days and injected with scopolamine (1mg/kg) for 7 days. We assessed the memory function using the Y-maze, novel object recognition, and passive avoidance tests. Protein expression analyses and histological analyses were performed using hippocampal tissues. YG treatment significantly restored cell viability against glutamate-induced apoptosis. It significantly suppressed glutamate-induced reactive oxygen species accumulation and mitochondrial dysfunction. It also increased Bcl-2 protein expression and decreased HO-1 protein expression. It activated the extracellular signal-regulated kinase/cAMP response element binding protein (ERK/CREB) signaling pathway and increased the expression of brain-derived neurotrophic factor (BDNF) under excitotoxic conditions. In the scopolamine-injected mice, YG ameliorated memory impairment in the Y-maze, novel object recognition, and passive avoidance tests; restored dysfunction in the acetylcholine, acetylcholinesterase expression levels; reduced neuronal damage in Nissl staining; and increased BDNF and phosphorylated ERK and CREB levels in Western blotting and immunofluorescence staining. Thus, YG exerted neuroprotective effects by activating ERK/CREB/BDNF signaling in the hippocampus, indicating its potential cognition-enhancing effects, especially in Alzheimer's disease.

Jing-an oral liquid alleviates Tourette syndrome via the NMDAR/MAPK/CREB pathway in vivo and in vitro

Context Jing-an oral liquid (JA) is a Chinese herbal formula used in the treatment of Tourette syndrome (TS); however, its mechanism is unclear. Objective To investigate the effects of JA on amino acid neurotransmitters and microglia activation in vivo and in vitro. Materials and methods Sixty male Sprague–Dawley rats were divided into a control group and 5 TS groups. TS was induced in rats with intraperitoneal injection of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (1 mg/kg) and in BV2 cells with lipopolysaccharide. Control and model rats were administered saline, whereas treatment groups were administered JA (5.18, 10.36, or 20.72 g/kg) or tiapride (a benzamide, 23.5 mg/kg) by gavage once daily for 21 days. Stereotypic behaviour was tested. The levels of N-methyl-d-aspartate receptor (NMDAR)/mitogen-activated protein kinase/cAMP response element-binding protein (CREB)-related proteins in striatum and BV2 cells were measured via western blots. CD11b and IBa1 levels were also measured. Ultra-high-performance liquid-chromatography was used to determine γ-aminobutyric acid (GABA), glutamic acid (Glu), and aspartic acid (ASP) levels. Results JA markedly alleviated the stereotype behaviour (25.92 ± 0.35 to 13.78 ± 0.47) in rats. It also increased NMDAR1 (0.48 ± 0.09 to 0.67 ± 0.08; 0.54 ± 0.07 to 1.19 ± 0.18) expression and down-regulated the expression of p-ERK, p-JNK, p-P38, and p-CREB in BV2 cells and rat striatum. Additionally, Glu, ASP, GABA, CD11b, and IBa1 levels were significantly decreased by JA. Discussion and conclusions JA suppressed microglia activation and regulated the levels of amino acid neurotransmitters, indicating that it could be a promising therapeutic agent for TS.

Cannabidiol attenuates methamphetamine-induced cardiac inflammatory response through the PKA/CREB pathway in rats.

BackgroundMethamphetamine (MA) abuse is a major global public health problem. However, it is not yet known whether cannabidiol (CBD) has protective effects on MA-induced cardiotoxicity. The present study investigated whether CBD has protective effects on MA-induced cardiac damage in rats via the protein kinase A/cyclic adenosine monophosphate (cAMP)-response element-binding protein (PKA/CREB) pathway.MethodsA total of 30 rats were randomly divided into 5 groups. The rats were administered MA (10 mg/kg) by intraperitoneal (IP) injection once a day for 4 weeks, and with CBD (40 or 80 mg/kg, IP) treatment 1 h before the MA injections. Morphological changes were determined using hematoxylin and eosin and Masson’s trichrome staining. The serum levels of interleukin (IL)-6 and IL-10 were detected using enzyme-linked immunoassay kits. The protein expression levels of cardiac troponin I (cTnI), PKA, phospho-PKA (p-PKA), CREB, and phospho-CREB (p-CREB) in the myocardium were detected by Western blot analysis.ResultsThere was no significant difference in body weight among the groups. Heart weight and the heart-to-body weight ratio were higher in the MA group than the control group, while CBD (80 mg/kg) pretreatment (CBD80 + MA group) reduced the heart weight and the heart-to-body weight ratio compared to the MA group. The chronic administration of MA resulted in a cardiac inflammatory response, the progressive development of fibrosis, and necrosis, while CBD treatment attenuated these lesions. The protein expression levels of PKA, p-PKA, CREB, and p-CREB increased following MA administration, but significantly decreased with CBD treatment. These results indicate that chronic MA administration leads to cardiotoxicity, but these effects can be attenuated by CBD pretreatment.ConclusionsThis study was the first to examine the protective effects of CBD on cardiotoxicity elicited by chronic MA exposure in rats. Our research suggests that CBD attenuates the cardiac inflammatory response induced by MA through the PKA/CREB pathway, and CBD may have potential clinical application in the treatment of MA-induced cardiotoxicity.

Correlations of ALD, Keap-1, and FoxO4 expression with traditional tumor markers and clinicopathological characteristics in colorectal carcinoma.

Aldolase A (A-2) (ALD), Kelch-like-ECH associated protein-1 (Keap-1), and Forkhead box O4 (FoxO4) are key regulatory proteins, which have been proven to be involved in tumor development. However, the clinicopathological significance of ALD, Keap-1, and FoxO4 expressions in colorectal (colon) carcinoma (CRC) is not clearly known. We sought to explore the clinicopathological significance of ALD, Keap-1, and FoxO4 in CRC to provide evidences for potential monitoring index of CRC. Cases of 199 CRC patients were analyzed retrospectively. Evaluation of ALD, cAMP response element-binding protein-2, cyclo-oxygenase 2, FoxO4, Keap-1, and p53 expressions in CRC patients was accomplished with immunohistochemical technique. The patients were divided into negative and positive groups in accordance with immunohistochemical result. We compared the clinicopathological characteristics of the patients in the 2 groups, coupled with analysis of the relationship between 6 aforesaid proteins and clinicopathological characteristics. Herein, we confirmed the association of tumor location with the expression of ALD, Keap-1, and FoxO4. Also, tumor differentiation was observed to associate significantly with the expression of Keap-1, FoxO4, and Cox-2. The data also revealed that there was a correlation between smoking and expression of ALD, Keap-1, FoxO4, p53, and Cox-2. Nevertheless, insignificant difference was observed when clinicopathological characteristics were compared with cAMP response element-binding protein-2 expression. These findings suggest that ALD, Keap-1, and FoxO4 reinvolved in CRC development, and thus may be considered as potential monitoring protein for CRC.

CREB-associated glycosylation and function in human disease.

The shortcomings of mRNA sequencing in explaining biological functions have resulted in proteomics gradually becoming a hotspot for research. However, the function of proteins becomes complicated as a result of post-translational modifications (PTMs) such as phosphorylation, glycosylation, acetylation, etc. Post-translational modifications do not change the physicochemical properties such as charge and solubility of the proteins, but they can have significant consequences on disease initiation in living organisms. The cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is an important transcription regulator in eukaryotic cells. It is involved in the development of neurodegenerative diseases, diabetic complications, tumorigenesis, and neurogenesis. Previously, researchers have paid much more attention to the phosphorylation modification of CREB. However, it seems that the functional regulation-mediated glycosylation modification of CREB was just beginning to be understood. In this review, the current studies and most updated insights on how the glycosylation modification of CREB affects targeted gene expression and disease development will be comprehensively discussed. We hope to further evaluate the role of CREB glycosylation on the regulation of gene function.

Head-tail-head neural wiring underlies gut fat storage in Caenorhabditis elegans temperature acclimation

Animals maintain the ability to survive and reproduce by acclimating to environmental temperatures. We showed here that Caenorhabditis elegans exhibited temperature acclimation plasticity, which was regulated by a head-tail-head neural circuitry coupled with gut fat storage. After experiencing cold, C. elegans individuals memorized the experience and were prepared against subsequent cold stimuli. The cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) regulated temperature acclimation in the ASJ thermosensory neurons and RMG head interneurons, where it modulated ASJ thermosensitivity in response to past cultivation temperature. The PVQ tail interneurons mediated the communication between ASJ and RMG via glutamatergic signaling. Temperature acclimation occurred via gut fat storage regulation by the triglyceride lipase ATGL-1, which was activated by a neuropeptide, FLP-7, downstream of CREB. Thus, a head-tail-head neural circuit coordinated with gut fat influenced experience-dependent temperature acclimation.

Maladaptive positive feedback production of ChREBPβ underlies glucotoxic β-cell failure

Preservation and expansion of β-cell mass is a therapeutic goal for diabetes. Here we show that the hyperactive isoform of carbohydrate response-element binding protein (ChREBPβ) is a nuclear effector of hyperglycemic stress occurring in β-cells in response to prolonged glucose exposure, high-fat diet, and diabetes. We show that transient positive feedback induction of ChREBPβ is necessary for adaptive β-cell expansion in response to metabolic challenges. Conversely, chronic excessive β-cell-specific overexpression of ChREBPβ results in loss of β-cell identity, apoptosis, loss of β-cell mass, and diabetes. Furthermore, β-cell “glucolipotoxicity” can be prevented by deletion of ChREBPβ. Moreover, ChREBPβ-mediated cell death is mitigated by overexpression of the alternate CHREBP gene product, ChREBPα, or by activation of the antioxidant Nrf2 pathway in rodent and human β-cells. We conclude that ChREBPβ, whether adaptive or maladaptive, is an important determinant of β-cell fate and a potential target for the preservation of β-cell mass in diabetes.

Overexpression of GhABF3 increases cotton(Gossypium hirsutum L.) tolerance to salt and drought

BackgroundPlants suffer from various abiotic stresses during their lifetime, of which drought and salt stresses are two main factors limiting crop yield and quality. Previous studies have shown that abscisic acid (ABA) responsive element binding protein (AREB)/ ABRE binding factors (ABFs) in bZIP transcription factors are involved in plant stress response in an ABA-dependent manner. However, little is known about the properties and functions of AREB/ABFs, especially ABF3, in cotton.ResultsHere, we reported the cloning and characterization of GhABF3. Expression of GhABF3 was induced by drought,salt and ABA treatments. Silencing of GhABF3 sensitized cotton to drought and salt stress, which was manifested in decreased cellular antioxidant capacity and chlorophyll content. Overexpression of GhABF3 significantly improved the drought and salinity tolerance of Arabidopsis and cotton. Exogenous expression of GhABF3 resulted in longer root length and less leaf wilting under stress conditions in Arabidopsis thaliana. Overexpressing GhABF3 significantly improved salt tolerance of upland cotton by reducing the degree of cellular oxidation, and enhanced drought tolerance by decreasing leaf water loss rate. The increased expression of GhABF3 up-regulated the transcriptional abundance of downstream ABA-inducible genes under salt stress in Arabidopsis.ConclusionIn conclusion, our results demonstrated that GhABF3 plays an important role in plant drought and salt tolerance. Manipulation of GhABF3 by biotechnology might be an important strategy to alter the stress resistance of cotton.

Dimethyl Itaconate Reduces α-MSH-Induced Pigmentation via Modulation of AKT and p38 MAPK Signaling Pathways in B16F10 Mouse Melanoma Cells.

Dimethyl itaconate (DMI) exhibits an anti-inflammatory effect. Activation of nuclear factor erythroid 2-related factor 2 (NRF2) is implicated in the inhibition of melanogenesis. Therefore, DMI and itaconic acid (ITA), classified as NRF2 activators, have potential uses in hyperpigmentation reduction. The activity of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), an important transcription factor for MITF gene promoter, is regulated by glycogen synthase kinase 3β (GSK3β) and protein kinase A (PKA). Here, we investigated the inhibitory effect of ITA and DMI on alpha-melanocyte-stimulating hormone (α-MSH)-induced MITF expression and the modulatory role of protein kinase B (AKT) and GSK3β in melanogenesis in B16F10 mouse melanoma cells. These cells were incubated with α-MSH alone or in combination with ITA or DMI. Proteins were visualized and quantified using immunoblotting and densitometry. Compared to ITA, DMI treatment exhibited a better inhibitory effect on the α-MSH-induced expression of melanogenic proteins such as MITF. Our data indicate that DMI exerts its anti-melanogenic effect via modulation of the p38 mitogen-activated protein kinase (MAPK) and AKT signaling pathways. In conclusion, DMI may be an effective therapeutic agent for both inflammation and hyperpigmentation.

Abstract 3499: M1069 as dual A2A/A2B adenosine receptor antagonist counteracts immune-suppressive mechanisms of adenosine and reduces tumor growth in vivo

Abstract Under physiological conditions, the extracellular concentrations of adenosine are low, however, levels dramatically increase under metabolically stressful conditions, including inflammation and cancer. The regulatory functions of adenosine are mediated through four members of the adenosine receptor family: A1, A2A, A2B, and A3. While A2A was considered the major contributor to adenosine-mediated suppression of T cell, natural killer cell, and myeloid cell functions, A2B has also recently emerged as a potential modulator of these processes. Both A2A and A2B signal through the same Gs-mediated activation of adenylate cyclase, which can allow the lower affinity A2B receptor to compensate for the inhibition of A2A in an adenosine-rich tumor microenvironment (TME). A2B receptors are also reported to support tumor growth independent of A2A through the Gq subunit of G-protein-coupled receptor-mediated production of vascular endothelial growth factor (VEGF) by myeloid and tumor cells. Therefore, simultaneous targeting of both the A2A and A2B receptors may provide a higher potential for cancer immunotherapy in an adenosine-rich TME, where A2B can act in compensatory or complementary means to A2A. M1069 is a small-molecule, dual antagonist of the A2A and A2B adenosine receptors with a selectivity of >100 fold against the A1 and A3 receptors. In assays with primary human T cells, M1069 caused a dose-dependent suppression of 5′-N-ethylcarboxamide adenosine (stable analog of adenosine)-stimulated cyclic adenosine monophosphate (cAMP) and phosphorylated cAMP- response element binding protein (pCREB) induction and rescue of interleukin (IL)-2 production (A2A readout). M1069 also suppressed VEGF production from human macrophages (A2B readout) in adenosine-rich settings. M1069 exhibited superior suppression of protumorigenic cytokine secretion, including CXCL1, CXCL5 and granulocyte-colony stimulating factor, and the rescue of IL12 secretion from adenosine-differentiated dendritic cells, as compared to an A2Aselective antagonist. In addition, in a one-way mixed lymphocyte reaction assay, adenosine-differentiated dendritic cells treated with M1069 demonstrated superior T cell activation compared to adenosine-differentiated dendritic cells treated with an A2A-selective antagonist. These findings were further corroborated with the results from in vivo studies in a murine CD73hi/adenosine-rich 4T1 syngeneic breast tumor model, in which M1069, but not an A2A-selective antagonist, reduced tumor growth as a monotherapy and enhanced anti-tumor activity with chemotherapeutic agents. In summary, M1069 is a potent, dual A2A/A2B adenosine receptor antagonist, which is expected to counteract immune-suppressive mechanisms in the presence of high concentrations of adenosine and enhance the anti-tumor activity of chemotherapies. Citation Format: Rinat Zaynagetdinov, Kai Schiemann, Kalyan Nallaparaju, Natalya Belousova, Armine Matevossian, Zhouxiang Chen, Giorgio Kradjian, Meghana Pandya, Nemisha Dawra, Eva-Maria Krauel, Elissaveta Petrova, Oliver Poeschke, David Fischer, Marc Lecomte, Andree Blaukat, Bayard Huck, Jacques Moisan. M1069 as dual A2A/A2B adenosine receptor antagonist counteracts immune-suppressive mechanisms of adenosine and reduces tumor growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3499.

Follicle stimulating hormone promotes production of renin through its receptor in juxtaglomerular cells of kidney

BackgroundPost-menopausal hypertension has been attributed solely to declining estrogen levels. The purpose of the research is to elucidate the mechanism by which follicle stimulating hormone(FSH) increases renin production involved in the regulation of blood pressure.MethodsThe expression of follicle stimulating hormone receptors (FSHRs) in renal juxtaglomerular cells and a As4.1 juxtaglomerular mouse cell line was evaluated. We established a mouse model by ovariectomy (OVX). Ovariectomized mice were treated with gonadotropin-releasing hormone agonist (GnRHa) (OVX + GnRHa). Ovariectomized mice initially received physiological doses of estrogen and were then injected with recombinant FSH (OVX + E + FSH).ResultsWe found that FSHR was expressed in mouse renal juxtaglomerular cells labeled by renin antibody and in As4.1 cells. FSH promoted renin synthesis via Gsα-coupled FSHRs that activated protein kinase A, cyclic adenosine monophosphate(cAMP) response element-binding protein, extracellular signal-regulated kinase (Erk1/2), Protein kinase B(AKT), and c-Jun N-terminal kinase signaling pathways in As4.1 cells. We found increased serum FSH levels in the ovariectomized mouse with concurrent increases in renin, angiotensin II, heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial blood pressure (MAP). Additionally, increases in serum renin, angiotensin II, HR, SBP, DBP, and MAP were reduced by the additional injection of GnRHa. Exogenous FSH administration completely reversed decreases in renin, angiotensin II, HR, SBP, DBP, and MAP even in mice that received physiological doses of estrogen to maintain normal estradiol levels.ConclusionsElevated FSH stimulates renin production involving a mechanism that may be relevant to the expression of FSH receptors in renal juxtaglomerular cells.

Cerebral pCREB-dependent social behavioral adversities following a short-term exposure to obesogenic diets in young hamsters

Excessive fat and sugar intake represents a risk towards the development of different pathologies, such as obesity, diabetes, sociability and memory deficits. Although the adolescence stage is a susceptible period for these and other risks, effects of energy-dense nutrients in such an age period have not been fully investigated. In the present study, neurobehavioral alterations following a 4-week exposure to either normal diet (ND) or high-fat diet (HFD) plus normal water (NW) or liquid sugar (LS) were evaluated in young hamsters. HFD + LS and ND + LS significantly reduced food intake and water consumption, which was, in the latter group, almost completely substituted by LS. All obesogenic diets accounted for increased abdominal fat and liver weight with respect to body weight (p < 0.05–0.001). Additionally, glucose levels notably increased (p < 0.0001) together with insulin and triglycerides in HFD + LS (p < 0.001) and ND + LS (p < 0.01) while cholesterol displayed only a moderate increase (p < 0.05) in HFD + NW and HFD + LS. Animals fed with HFD and/or LS exhibited impaired social memory plus increased winning percentages (0.05 < p < 0.01) during the tube test. Interestingly, these same treatments led to a down-regulation of phosphorylated cAMP Response-Element Binding Protein (pCREB) in HFD + NW (p < 0.0001) for all areas, but rather was upregulated (p < 0.05) in ND + LS of the amygdala. Overall, in view of a brief exposure to palatable foods interfering with normal metabolic and social memory activities, the downregulation of pCREB constitutes a key indicator of neurobehavioral deficits during obesogenic diets. Compensatory mechanisms may be also occurring in the amygdala that strongly regulates emotional states via connections with other limbic areas.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Unveiling OASIS family as a key player in hypoxia\u2013ischemia cases induced by cocaine using generative adversarial networks

Repeated cocaine use poses many serious health risks to users. One of the risks is hypoxia and ischemia (HI). To restore the biological system against HI, complex biological mechanisms operate at the gene level. Despite the complexity of biological mechanisms, there are common denominator genes that play pivotal roles in various defense systems. Among these genes, the cAMP response element-binding (Creb) protein contributes not only to various aspects of drug-seeking behavior and drug reward, but also to protective mechanisms. However, it is still unclear which Creb members are key players in the protection of cocaine-induced HI conditions. Herein, using one of the state-of-the-art deep learning methods, the generative adversarial network, we revealed that the OASIS family, one of the Creb family, is a key player in various defense mechanisms such as angiogenesis and unfolded protein response against the HI state by unveiling hidden mRNA expression profiles. Furthermore, we identified mysterious kinases in the OASIS family and are able to explain why the prefrontal cortex and hippocampus are vulnerable to HI at the genetic level.

Fraxinol Stimulates Melanogenesis in B16F10 Mouse Melanoma Cells through CREB/MITF Signaling.

Melanin pigment produced in melanocytes plays a protective role against ultraviolet radiation. Selective destruction of melanocytes causes chronic depigmentation conditions such as vitiligo, for which there are very few specific medical treatments. Here, we found that fraxinol, a natural coumarin from Fraxinus plants, effectively stimulated melanogenesis. Treatment of B16-F10 cells with fraxinol increased the melanin content and tyrosinase activity in a concentration-dependent manner without causing cytotoxicity. Additionally, fraxinol enhanced the mRNA expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2. Fraxinol also increased the expression of microphthalmia-associated transcription factor at both mRNA and protein levels. Fraxinol upregulated the phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). Furthermore, H89, a cAMP–dependent protein kinase A inhibitor, decreased fraxinol-induced CREB phosphorylation and microphthalmia-associated transcription factor expression and significantly attenuated the fraxinol-induced melanin content and intracellular tyrosinase activity. These results suggest that fraxinol enhances melanogenesis via a protein kinase A-mediated mechanism, which may be useful for developing potent melanogenesis stimulators.

Anti-acne effects of Castanea crenata bur extract and identification of active compound

Acne vulgaris is a common disease of the pilosebaceous unit. Hyperseborrhea, a follicular colonization by Cutibacterium acnes and a complex inflammatory state are pathogenic factors of acne vulgaris. In the present study we investigated the anti-acne efficacy of Castanea crenata bur extract (CBE) in vitro and searched active compound for mitigating hyperseborrhea. In sebocytes, CBE inhibited the sebum synthesis through downregulation of sterol response element-binding protein-1 and peroxisome proliferator-activated receptor γ expression. CBE also inhibited the 5-alpha reductase activity which is associated with androgen-induced sebum production. Moreover, CBE showed anti-inflammatory effect in C. acnes and free fatty acid-induced inflammatory condition through suppressing Toll-like receptor 2 activity. Anti-inflammatory effect was also observed in keratinocytes via inhibition of NF-κB translocation into nuclei. Finally, we identified the ellagic acid as an active compound for inhibiting sebum production in CBE. These findings suggest that CBE have potential to be a multi-target agent for acne vulgaris and a good source of ellagic acid as an anti-sebum compound.

Punicalagin and Ketogenic Amino Acids Loaded Organic Lipid Carriers Enhance the Bioavailability, Mitochondrial β-Oxidation, and Ketogenesis in Maturing Adipocytes.

The identification of lipolytic bioactive compounds via the functional stimulation of carbohydrate response element-binding protein-1 (CREBp-1) and AMP-activated protein kinase (AMPK) is most warranted. Nano lipid carriers (NLCs) are currently being considered within drug delivery development as they facilitate controlled drug release and have intracellular bioavailability after encapsulating the active principles with lipid matrix. The present study has been designed to synthesize punicalagin, and ketogenic amino acids (KAA) loaded with organic lipid carriers to optimize the liposome-assisted intracellular delivery’s bioavailability. Punicalagin (PUNI) and KAA (tryptophan, methionine, threonine, lysine, and leucine) were encapsulated with chia seed phospholipids by homogenization, emulsification, and cold ultra-sonication method to obtain nano lipid carriers (NLC). The physicochemical characterization of NLCs has been carried out using Zetasizer, FT-IR, and TEM analysis. Punicalagin and ketogenic amino acid-loaded NLCs (NLC-PUNI-KAA) were identified with an average diameter of 240 to 800 nm. The biosafety of NLC-PUNI-KAA has been evaluated in human mesenchymal stem cells. PI staining confirmed that a 0.4, 0.8 or 1.6μg/dL dose of NLC-PUNI-KAA potentially maintains nuclear integration. NLC-PUNI-KAA treated with maturing adipocytes decreased lipid accumulation and significantly increased the gene expression levels of fatty acid beta-oxidation (PPARγC1α, UCP-1 and PRDM-16) pathways when compared to free PUNI (5 μg/dL) treatment. The lipolytic potential has been confirmed by the functional activation of AMPK and CREBp-1 protein levels. In conclusion, NLC-PUNI-KAA treatment effectively increased mitochondrial efficiency more than free punicalagin or orlistat treated maturing adipocyte. Enhanced lipolysis and decreased hypertrophic adipocyte resulted in decreased adipokine secretion, which has been associated with the suppression of obesity-associated comorbidities and vascular cell inflammation. The bioefficacy and lipolytic potential of water-soluble punicalagin have been improved after functional modification into NLCs.

Intricate coupling between the transactivation and basic-leucine zipper domains governs phosphorylation of transcription factor ATF4 by casein kinase 2

Most transcription factors possess at least one long intrinsically disordered transactivation domain that binds to a variety of coactivators and corepressors and plays a key role in modulating the transcriptional activity. Despite the crucial importance of these domains, the structural and functional basis of transactivation remains poorly understood. Here, we focused on activating transcription factor 4 (ATF4)/cAMP response element-binding protein-2, an essential transcription factor for cellular stress adaptation. Bioinformatic sequence analysis of the ATF4 transactivation domain sequence revealed that the first 125 amino acids have noticeably less propensity for structural disorder than the rest of the domain. Using solution nuclear magnetic resonance spectroscopy complemented by a range of biophysical methods, we found that the isolated transactivation domain is predominantly yet not fully disordered in solution. We also observed that a short motif at the N-terminus of the transactivation domain has a high helical propensity. Importantly, we found that the N-terminal region of the transactivation domain is involved in transient long-range interactions with the basic-leucine zipper domain involved in DNA binding. Finally, in vitro phosphorylation assays with the casein kinase 2 show that the presence of the basic-leucine zipper domain is required for phosphorylation of the transactivation domain. This study uncovers the intricate coupling existing between the transactivation and basic-leucine zipper domains of ATF4, highlighting its potential regulatory significance.

Bone morphogenic protein 9 is a novel thermogenic hepatokine secreted in response to cold exposure

ObjectiveMaintaining a constant core body temperature is essential to homeothermic vertebrate survival. Adaptive thermogenesis in brown adipose tissue and skeletal muscle is the primary mechanism of adjustment to an external stimulus such as cold exposure. Recently, several reports have revealed that the liver can play a role as a metabolic hub during adaptive thermogenesis. In this study, we suggest that the liver plays a novel role in secreting thermogenic factors in adaptive thermogenesis. Bone morphogenetic protein 9 (BMP9) is a hepatokine that regulates many biological processes, including osteogenesis, chondrogenesis, hematopoiesis, and angiogenesis. Previously, BMP9 was suggested to affect preadipocyte proliferation and differentiation. However, the conditions and mechanisms underlying hepatic expression and secretion and adipose tissue browning of BMP9 remain largely unknown. In this study, we investigated the physiological conditions for secretion and the regulatory mechanism of hepatic Bmp9 expression and the molecular mechanism by which BMP9 induces thermogenic gene program activation in adipose tissue. Here, we also present the pharmacological effects of BMP9 on a high-fat-induced obese mouse model. MethodsTo investigate the adaptive thermogenic role of BMP9 in vivo, we challenged mice with cold temperature exposure for 3 weeks and then examined the BMP9 plasma concentration and hepatic expression level. The cellular mechanism of hepatic Bmp9 expression under cold exposure was explored through promoter analysis. To identify the role of BMP9 in the differentiation of brown and beige adipocytes, we treated pluripotent stem cells and inguinal white adipose tissue (iWAT)-derived stromal-vascular (SV) cells with BMP9, and brown adipogenesis was monitored by examining thermogenic gene expression and signaling pathways. Furthermore, to evaluate the effect of BMP9 on diet-induced obesity, changes in body composition and glucose tolerance were analyzed in mice administered recombinant BMP9 (rBMP9) for 8 weeks. ResultsHepatic Bmp9 expression and plasma levels in mice were significantly increased after 3 weeks of cold exposure. Bmp9 mRNA expression in the liver was regulated by transcriptional activation induced by cAMP response-element binding protein (CREB) and CREB-binding protein (CBP) on the Bmp9 promoter. Treatment with BMP9 promoted the differentiation of multipotent stem cells and iWAT-derived SV cells into beige adipocytes, as indicated by the increased expression of brown adipocyte and mitochondrial biogenesis markers. Notably, activation of the mothers against decapentaplegic homolog 1 (Smad1) and p44/p42 mitogen-activated protein kinase (MAPK) pathways was required for the induction of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression in BMP9-induced differentiation of SVs into beige adipocytes. The administration of rBMP9 in vivo also induced browning markers in white adipose tissue. In high-fat diet-induced obese mice, rBMP9 administration conferred protection against obesity and enhanced glucose tolerance. ConclusionsBMP9 is a hepatokine regulated by cold-activated CREB and CBP and enhances glucose and fat metabolism by promoting the activation of the thermogenic gene program in adipocytes. These data implicate BMP9 as a potential pharmacological tool for protecting against obesity and type 2 diabetes.