Phosphorylation Of cAMP Response Element-binding Protein Research Articles

Angiotensin II participates in mitochondrial thermogenic functions via the activation of glycolysis in chemically induced human brown adipocytes

Brown adipocytes are potential therapeutic targets for the prevention of obesity-associated metabolic diseases because they consume circulating glucose and fatty acids for heat production. Angiotensin II (Ang II) peptide is involved in the pathogenesis of obesity- and cold-induced hypertension; however, the mechanism underlying the direct effects of Ang II on human brown adipocytes remains unclear. Our transcriptome analysis of chemical compound-induced brown adipocytes (ciBAs) showed that the Ang II type 1 receptor (AGTR1), but not AGTR2 and MAS1 receptors, was expressed. The Ang II/AGTR1 axis downregulated the expression of mitochondrial uncoupling protein 1 (UCP1). The simultaneous treatment with β-adrenergic receptor agonists and Ang II attenuated UCP1 expression, triglyceride lipolysis, and cAMP levels, although cAMP response element-binding protein (CREB) phosphorylation was enhanced by Ang II mainly through the protein kinase C pathway. Despite reduced lipolysis, both coupled and uncoupled mitochondrial respiration was enhanced in Ang II-treated ciBAs. Instead, glycolysis and glucose uptake were robustly activated upon treatment with Ang II without a comprehensive transcriptional change in glucose metabolic genes. Elevated mitochondrial energy status induced by Ang II was likely associated with UCP1 repression. Our findings suggest that the Ang II/AGTR1 axis participates in mitochondrial thermogenic functions via glycolysis.

Anti-Melanogenic Effects of Takifugu flavidus Muscle Hydrolysate in B16F10 Melanoma Cells and Zebrafish

Abnormal melanogenesis can lead to hyperpigmentation. Tyrosinase (TYR), a key rate-limiting enzyme in melanin production, is an important therapeutic target for these disorders. We investigated the TYR inhibitory activity of hydrolysates extracted from the muscle tissue of Takifugu flavidus (TFMH). We used computer-aided virtual screening to identify a novel peptide that potently inhibited melanin synthesis, simulated its binding mode to TYR, and evaluated functional efficacy in vitro and in vivo. TFMH inhibited the diphenolase activities of mTYR, reducing TYR substrate binding activity and effectively inhibiting melanin synthesis. TFMH indirectly reduced cAMP response element-binding protein phosphorylation in vitro by downregulating melanocortin 1 receptor expression, thereby inhibiting expression of the microphthalmia-associated transcription factor, further decreasing TYR, tyrosinase related protein 1, and dopachrome tautomerase expression and ultimately impeding melanin synthesis. In zebrafish, TFMH significantly reduced black spot formation. TFMH (200 μg/mL) decreased zebrafish TYR activity by 43% and melanin content by 52%. Molecular dynamics simulations over 100 ns revealed that the FGFRSP (T-6) peptide stably binds mushroom TYR via hydrogen bonds and ionic interactions. T-6 (400 μmol/L) reduced melanin content in B16F10 melanoma cells by 71% and TYR activity by 79%. In zebrafish, T-6 (200 μmol/L) inhibited melanin production by 64%. TFMH and T-6 exhibit good potential for the development of natural skin-whitening cosmetic products.

Rosemary extract and rosmarinic acid accelerate elastic fiber formation by increasing the expression of elastic fiber components in dermal fibroblasts.

Ultraviolet (UV)-induced skin photoaging is caused by qualitative and quantitative degradation of dermal extracellular matrix components such as collagen and elastic fibers. Elastic fibers are important for maintaining cutaneous elasticity, despite their small amount in the skin. Previously, microfibril-associated protein 4 (MFAP-4), which is downregulated in photoaging dermis, has been found to be essential for elastic fiber formation by interaction with both fibrillin-1 and elastin, which are core components of elastic fiber. In addition, enhanced cutaneous MFAP-4 expression in a human skin-xenografted murine photoaging model protects against UV-induced photodamage accompanied by the prevention of elastic fiber degradation and aggravated elasticity. We therefore hypothesized that the upregulation of MFAP-4 in dermal fibroblasts may more efficiently accelerate elastic fiber formation. We screened botanical extracts for MFAP-4 expression-promoting activity in normal human dermal fibroblasts (NHDFs). We found that rosemary extract markedly promotes early microfibril formation and mature elastic fiber formation along with a significant upregulation of not only MFAP-4 but also fibrillin-1 and elastin in NHDFs. Furthermore, rosmarinic acid, which is abundant in rosemary extract, accelerated elastic fiber formation via upregulation of transforming growth factor β-1. This was achieved by the induction of cAMP response element-binding protein phosphorylation, demonstrating that rosmarinic acid represents one of the active ingredients in rosemary extract. Based on the findings in this study, we conclude that rosemary extract and rosmarinic acid represent promising materials that exert a preventive or ameliorative effect on skin photoaging by accelerating elastic fiber formation.

Brain regulates weight bearing bone through PGE2 skeletal interoception: implication of ankle osteoarthritis and pain

Bone is a mechanosensitive tissue and undergoes constant remodeling to adapt to the mechanical loading environment. However, it is unclear whether the signals of bone cells in response to mechanical stress are processed and interpreted in the brain. In this study, we found that the hypothalamus of the brain regulates bone remodeling and structure by perceiving bone prostaglandin E2 (PGE2) concentration in response to mechanical loading. Bone PGE2 levels are in proportion to their weight bearing. When weight bearing changes in the tail-suspension mice, the PGE2 concentrations in bones change in line with their weight bearing changes. Deletion of cyclooxygenase-2 (COX2) in the osteoblast lineage cells or knockout of receptor 4 (EP4) in sensory nerve blunts bone formation in response to mechanical loading. Moreover, knockout of TrkA in sensory nerve also significantly reduces mechanical load-induced bone formation. Moreover, mechanical loading induces cAMP-response element binding protein (CREB) phosphorylation in the hypothalamic arcuate nucleus (ARC) to inhibit sympathetic tyrosine hydroxylase (TH) expression in the paraventricular nucleus (PVN) for osteogenesis. Finally, we show that elevated PGE2 is associated with ankle osteoarthritis (AOA) and pain. Together, our data demonstrate that in response to mechanical loading, skeletal interoception occurs in the form of hypothalamic processing of PGE2-driven peripheral signaling to maintain physiologic bone homeostasis, while chronically elevated PGE2 can be sensed as pain during AOA and implication of potential treatment.

Caffeine-induced protein kinase A activation restores cognitive deficits induced by sleep deprivation by regulating O-GlcNAc cycling in adult zebrafish.

Sleep deprivation (SD) is widely acknowledged as a significant risk factor for cognitive impairment. In this study, intraperitoneal caffeine administration significantly ameliorated the learning and memory (L/M) deficits induced by SD and reduced aggressive behaviors in adult zebrafish. SD led to a reduction in protein kinase A (PKA) phosphorylation, phosphorylated-cAMP response element-binding protein (p-CREB), and c-Fos expression in zebrafish brain. Notably, these alterations were effectively reversed by caffeine. In addition, caffeine mitigated neuroinflammation induced by SD, as evident from suppression of the SD-mediated increase in glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) activation. Caffeine restored normal O-GlcNAcylation and O-GlcNAc transferase (OGT) levels while reversing the increased expression of O-GlcNAcase (OGA) in zebrafish brain after SD. Intriguingly, rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, effectively mitigated cognitive deficits, restored p-CREB and c-Fos levels, and attenuated the increase in GFAP in brain induced by SD. In addition, rolipram reversed the decrease in O-GlcNAcylation and OGT expression as well as elevation of OGA expression following SD. Treatment with H89, a PKA inhibitor, significantly impaired the L/M functions of zebrafish compared with the control group, inducing a decrease in O-GlcNAcylation and OGT expression and, conversely, an increase in OGA expression. The H89-induced changes in O-GlcNAc cycling and L/M dysfunction were effectively reversed by glucosamine treatment. H89 suppressed, whereas caffeine and rolipram promoted O-GlcNAc cycling in Neuro2a cells. Our collective findings underscore the interplay between PKA signaling and O-GlcNAc cycling in the regulation of cognitive function in the brain, offering potential therapeutic targets for cognitive deficits associated with SD.NEW & NOTEWORTHY Our observation highlights the intricate interplay between cAMP/PKA signaling and O-GlcNAc cycling, unveiling a novel mechanism that potentially governs the regulation of learning and memory functions. The dynamic interplay between these two pathways provides a novel and nuanced perspective on the molecular foundation of learning and memory regulation. These insights open avenues for the development of targeted interventions to treat conditions that impact cognitive function, including SD.

Secretin-dependent signals in the ventromedial hypothalamus regulate energy metabolism and bone homeostasis in mice

Secretin, though originally discovered as a gut-derived hormone, is recently found to be abundantly expressed in the ventromedial hypothalamus, from which the central neural system controls satiety, energy metabolism, and bone homeostasis. However, the functional significance of secretin in the ventromedial hypothalamus remains unclear. Here we show that the loss of ventromedial hypothalamus-derived secretin leads to osteopenia in male and female mice, which is primarily induced by diminished cAMP response element-binding protein phosphorylation and upregulation in peripheral sympathetic activity. Moreover, the ventromedial hypothalamus-secretin inhibition also contributes to hyperphagia, dysregulated lipogenesis, and impaired thermogenesis, resulting in obesity in male and female mice. Conversely, overexpression of secretin in the ventromedial hypothalamus promotes bone mass accrual in mice of both sexes. Collectively, our findings identify an unappreciated secretin signaling in the central neural system for the regulation of energy and bone metabolism, which may serve as a new target for the clinical management of obesity and osteoporosis.

A Capsicum annuum L. seed extract exerts anti-neuroexcitotoxicity in HT22 hippocampal neurons.

The hippocampal memory deficit stands out as a primary symptom in neurodegenerative diseases, including Alzheimer's disease. While numerous therapeutic candidates have been proposed, they primarily serve to delay disease progression. Given the irreversible brain atrophy or injury associated with these conditions, current research efforts are concentrated on preventive medicine strategies. Herein, we investigated whether the extracts of Capsicum annuum L. seeds (CSE) and Capsicum annuum L. pulp (CPE) have preventive properties against glutamate-induced neuroexcitotoxicity (one of the main causes of Alzheimer's disease) in HT22 hippocampal neuronal cells. Pretreatment with CSE demonstrated significant anti-neuroexcitotoxic activity, whereas CPE did not exhibit such effects. Specifically, CSE pretreatment dose-dependently inhibited the elevation of excitotoxic elements (intracellular calcium influx and reactive oxygen species; ROS) and apoptotic elements (p53 and cleaved caspase-3). In addition, the glutamate-induced alterations of neuronal activity indicators (brain-derived neurotrophic factor; BDNF and cAMP response element-binding protein phosphorylation; CREB) were significantly attenuated by CSE treatment. We also found that luteolin is the main bioactive compound corresponding to the anti-neuroexcitotoxic effects of CSE. Our results strongly suggest that Capsicum annuum L. seeds (but not its pulp) could be candidates for neuro-protective resources especially under conditions of neuroexcitotoxicity. Its underlying mechanisms may involve the amelioration of ROS-mediated cell death and BDNF-related neuronal inactivity and luteolin would be an active compound.

Bortezomib Is Toxic but Induces Neurogenesis and Inhibits TUBB3 Degradation in Rat Neural Stem Cells.

Bortezomib (BTZ) is a proteasome inhibitor used to treat multiple myeloma (MM). However, the induction of peripheral neuropathy is one of the major concerns in using BTZ to treat MM. In the current study, we have explored the effects of BTZ (0.01-5 nM) on rat neural stem cells (NSCs). BTZ (5 nM) induced cell death; however, the percentage of neurons was increased in the presence of mitogens. BTZ reduced the B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein ratio in proliferating NSCs and differentiated cells. Inhibition of βIII-tubulin (TUBB3) degradation was observed, but not inhibition of glial fibrillary acidic protein degradation, and a potential PEST sequence was solely found in TUBB3. In the presence of growth factors, BTZ increased cAMP response element-binding protein (CREB) phosphorylation, brain-derived neurotrophic factor (Bdnf) transcription, BDNF expression, and Tubb3 transcription in NSCs. However, in the neuroblastoma cell line, SH-SY5Y, BTZ (1-20 nM) only increased cell death without increasing CREB phosphorylation, Bdnf transcription, or TUBB3 induction. These results suggest that although BTZ induces cell death, it activates neurogenic signals and induces neurogenesis in NSCs.

Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer.

In castration-resistant prostate cancer (CRPC), increased glucocorticoid receptor (GR) expression and ensuing transcriptional activity have been proposed as an oncogenic "bypass" mechanism in response to androgen receptor (AR) signaling inhibition (ARSi). Here, we report that GR transcriptional activity acquired following ARSi is associated with the upregulation of cyclic adenosine monophosphate (cAMP)-associated gene expression pathways in both model systems and metastatic prostate cancer patient samples. In the context of ARSi, the expression of GR-mediated genes encoding cAMP signaling pathway-associated proteins can be inhibited by treatment with selective GR modulators (SGRMs). For example, in the context of ARSi, we found that GR activation resulted in upregulation of protein kinase inhibitor beta (PKIB) mRNA and protein levels, leading to nuclear accumulation of the cAMP-dependent protein kinase A catalytic subunit (PKA-c). Increased PKA-c, in turn, is associated with increased cAMP response element-binding protein phosphorylation and activity. Furthermore, enzalutamide and SGRM combination therapy in mice bearing CRPC xenografts delayed CRPC progression compared with enzalutamide therapy alone, and reduced tumor PKIB mRNA expression. Supporting the clinical importance of GR/PKA signaling activation in CRPC, we found a significant enrichment of both cAMP pathway signaling-associated gene expression and high NR3C1 (GR) activity in patient-derived xenograft models and metastatic human CRPC samples. These findings suggest a novel mechanism linking CRPC-induced GR transcriptional activity with increased cAMP signaling in AR-antagonized CRPC. Furthermore, our findings suggest that GR-specific modulation in addition to AR antagonism may delay GR+ CRPC time to recurrence, at least in part, by inhibiting tumor cAMP/PKA pathways.

[Baicalin improves inflammatory response of human microglia by regulating cAMP-PKA-NF-κB/CREB pathway].

This study aims to investigate the effects of baicalein(BAI) on lipopolysaccharide(LPS)-induced human microglial clone 3(HMC3) cells, with a focus on suppressing inflammatory responses and elucidating the potential mechanism underlying the therapeutic effects of BAI on ischemic stroke via modulating the cAMP-PKA-NF-κB/CREB pathway. The findings have significant implications for the application of traditional Chinese medicine in treating cerebral ischemic diseases. First, the safe dosage of BAI was screened, and then an inflammation model was established with HMC3 cells by induction with LPS for 24 h. The cells were assigned into a control group, a model group, and high-, medium-, and low-dose(5, 2.5, and 1.25 μmol·L~(-1), respectively) BAI groups. The levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in cell extracts, as well as the levels of interleukin-1β(IL-1β), IL-6, tumor necrosis factor-α(TNF-α), and cyclic adenosine monophosphate(cAMP) in the cell supernatant, were measured. Western blot was performed to determine the expression of protein kinase A(PKA), phosphorylated cAMP-response element binding protein(p-CREB), and nuclear factor-kappa B p65(NF-κB p65). Hoechst 33342/PI staining was employed to assess cell apoptosis. High and low doses of BAI were used for treatment in the research on the mechanism. The results revealed that BAI at the concentrations of 10 μmol·L~(-1) and below had no impact on normally cultured HMC3 cells. LPS induction at 200 ng·mL~(-1) for 24 h reduced the SOD activity and increased the MDA content in HMC3 cells. However, 5, 2.5, and 1.25 μmol·L~(-1) BAI significantly increased the SOD activity and 5 μmol·L~(-1) BAI significantly decreased the MDA content. In addition, BAI ameliorated the M1 polarization of HMC3 cells induced by LPS, as indicated by cellular morphology. The results of ELISA demonstrated that BAI significantly lowered the levels of TNF-α, IL-1β, IL-6, and cAMP in the cell supernatant. Western blot revealed that BAI up-regulated the protein levels of PKA and p-CREB while down-regulating the expression of NF-κB p65. Hoechst 33342/PI staining results indicated that BAI mitigated the apoptosis of HMC3 cells. Overall, the results indicated that BAI had protective effects on the HMC3 cells induced by LPS, and could inhi-bit inflammatory response and improve cell apoptosis, which might be related to the regulation of the cAMP-PKA-NF-κB/CREB pathway.

Gene Rearrangement and Expression of PRKACA and PRKACB Govern Morphobiology of Pancreatobiliary Oncocytic Neoplasms

Intraductal oncocytic papillary neoplasms (IOPNs) are distinct from intraductal papillary mucinous neoplasms based on characteristic morphologic and genetic features represented by fusion genes involving PRKACA or PRKACB (PRKACA/B). However, pancreatic and biliary tumors with partial oncocytic features are often encountered clinically, and their molecular features are yet to be clarified. This study included 80 intraductal papillary neoplasms: 32 tumors with mature IOPN morphology (typical), 28 with partial or subclonal oncocytic features (atypical), and 20 without oncocytic features (control). We analyzed PRKACA/B fusion genes, including ATP1B1::PRKACA, DNAJB1::PRKACA, and ATP1B1::PRKACB, by reverse-transcription PCR; mRNA expression of fusion genes and nonrearranged PRKACA/B genes by quantitative reverse-transcription PCR; mutations in KRAS, BRAF, and GNAS by targeted sequencing or droplet digital PCR; and the expression of cyclic adenosine monophosphate (cAMP)–dependent protein kinase catalytic subunits α (PRKACA) and β (PRKACB), phosphorylated cAMP response element-binding protein, and aberrations of p16, p53, SMAD4, STK11, and β-catenin by immunohistochemistry. PRKACA/B fusion genes were detected in 100% (32/32) of typical, 46% (13/28) of atypical, and 0% (0/20) of control (P < .05). Expression of PRKACA, PRKACB, and phosphorylated cAMP response element-binding protein was upregulated in neoplasms with PRKACA/B fusion genes (P < .05). mRNA expression of the PRKACA/B fusion genes and protein expression of PRKACA or PRKACB tended to be higher in typical than in atypical cases (mRNA, P = .002; protein expression, P = .054). In some atypical neoplasms with mixed subtypes, PRKACA/B fusion genes were superimposed exclusively on oncocytic components. Typical IOPNs harbored fewer KRAS and GNAS mutations than control samples and fewer alterations in p53 and STK11 than atypical samples (P < .05). In conclusion, PRKACA/B fusion genes not only are the characteristic drivers of IOPNs but also play a crucial role in the development of subclonal oncocytic neoplasms. Moreover, oncocytic morphology is strongly associated with upregulation of PRKACA/B, which may provide clues for potential therapeutic options.

CRH-R2 signalling modulates feeding and circadian gene expression in hypothalamic mHypoA-2/30 neurons.

The hypothalamic type 2 corticotropin releasing hormone receptor (CRH-R2) plays critical roles in homeostatic regulation, particularly in fine tuning stress recovery. During acute stress, the CRH-R2 ligands CRH and urocortins promote adaptive responses and feeding inhibition. However, in rodent models of chronic stress, over-exposure of hypothalamic CRH-R2 to its cognate agonists is associated with urocortin 2 (Ucn2) resistance; attenuated cAMP-response element binding protein (CREB) phosphorylation and increased food intake. The molecular mechanisms involved in these altered CRH-R2 signalling responses are not well described. In the present study, we used the adult mouse hypothalamus-derived cell line mHypoA-2/30 to investigate CRH-R2 signalling characteristics focusing on gene expression of molecules involved in feeding and circadian regulation given the role of clock genes in metabolic control. We identified functional CRH-R2 receptors expressed in mHypoA-2/30 cells that differentially regulate CREB and AMP-activated protein kinase (AMPK) phosphorylation and downstream expression of the appetite-regulatory genes proopiomelanocortin (Pomc) and neuropeptide Y (Npy) in accordance with an anorexigenic effect. We studied for the first time the effects of Ucn2 on clock genes in native and in a circadian bioluminescence reporter expressing mHypoA-2/30 cells, detecting enhancing effects of Ucn2 on mRNA levels and rhythm amplitude of the circadian regulator Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), which could facilitate anorexic responses in the activity circadian phase. These data uncover novel aspects of CRH-R2 hypothalamic signalling that might be important in regulation of circadian feeding during stress responses.

Magnolia officinalis bark extract improves depressive-like behavior in DSS-induced colitis mice

Most studies on the pathogenesis of inflammatory bowel disease (IBD) focus on gut function damage and gastrointestinal symptoms; however, psychiatric disorders, including depression and anxiety, have also been recognized as significant causes. Recently, we reported that Magnolia officinalis bark extract (MBE) alleviated enterocyte death in dextran sulfate sodium (DSS)-induced colitis mouse model that mimics IBD; however, the therapeutic effects of MBE on neurological symptoms and underlying mechanisms of IBD remain unclear. In this study, we aimed to elucidate the mechanisms by which microglia and astrocytes improve MBE-induced depression and anxiety. We found that MBE treatment improved DSS-induced weight loss, colon-length shortening, and cell death, with ameliorateon of depression/anxiety-like behavioral disorders. Systemic treatment with MBE significantly decreased DSS-induced proliferation of microglia and astrocytes in the hippocampal region, and attenuated the increase in pro-inflammatory cytokines, including IL-6 and COX-2. Interestingly, MBE alleviated DSS-induced alterations of the serotonergic system by improving serotonin levels, and expression of serotonin 4 receptor (5-HT4R) and cAMP response element-binding protein (CREB) phosphorylation in the hippocampus. Overall, our findings indicate that MBE exerts protective effects not only on gastrointestinal function but also on neuropsychiatric disorders caused by colitis, which may be developed to treat IBD.

Sargassum fusiforme Extract Induces Melanogenesis through the cAMP/PKA/CREB Signaling Pathway

The aim of this study was to investigate the effect of Sargassum fusiforme extract (SFE) on melanogenesis and its mechanism both in vitro and ex vivo. The melanogenic-inducing effect of SFE was evaluated using a melanin contents assay and a cellular tyrosinase activity assay. To investigate whether SFE could protect melanocytes against oxidative stress, hydrogen peroxidase was used. The molecular mechanism underlying the effect of SFE on melanogenesis was determined via Western blot analysis of tyrosinase, a microphthalmia-associated transcription factor (MITF), and a phosphorylated cAMP response element-binding protein (p-CREB) expression. The degree of pigmentation in a 3D skin model was determined by measuring the L* values. Contents of melanin in ex vivo human hair follicles were evaluated via Fontana–Masson staining. SFE significantly increased melanin contents and cellular tyrosinase activity in human epidermal melanocytes. SFE also increased the phosphorylation of CREB and the protein levels of tyrosinase and MITF. Moreover, SFE attenuated oxidative stress-induced cytotoxicity and depigmentation. Finally, the melanogenesis promoting effect of SFE was confirmed in both a 3D skin model and ex vivo human hair follicles. These findings suggest that SFE can induce melanogenesis via the cAMP/PKA/CREB signaling pathway in human epidermal melanocytes through its hyperpigmentation activity.

Unleashing T cell anti-tumor immunity: new potential for 5-Nonloxytryptamine as an agent mediating MHC-I upregulation in tumors

BackgroundNew therapies are urgently needed in melanoma, particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors. To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen and identified 5-Nonyloxytryptamine (5-NL), a serotonin agonist, as increasing the ability of T cells to target tumor cells.MethodsThe pharmacological screen utilized lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells and melanoma B16.F10 cells expressing the LCMV gp33 CTL epitope. In vivo tumor growth in C57BL/6 J and NSG mice, in vivo antibody depletion, flow cytometry, immunoblot, CRISPR/Cas9 knockout, histological and RNA-Seq analyses were used to decipher 5-NL’s immunomodulatory effects in vitro and in vivo.Results5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts’ immune system, specifically CD8+ T cells. 5-NL’s pro-immune effects were not directly consequential to T cells. Rather, 5-NL upregulated antigen presenting machinery in melanoma and other tumor cells in vitro and in vivo without increasing PD-L1 expression. Mechanistic studies indicated that 5-NL’s induced MHC-I expression was inhibited by pharmacologically preventing cAMP Response Element-Binding Protein (CREB) phosphorylation. Importantly, 5-NL combined with anti-PD1 therapy showed significant improvement when compared to single anti-PD-1 treatment.ConclusionsThis study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors.

Alcohol as a Modifiable Risk Factor for Alzheimer's Disease-Evidence from Experimental Studies.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive impairment and memory loss. Epidemiological evidence suggests that heavy alcohol consumption aggravates AD pathology, whereas low alcohol intake may be protective. However, these observations have been inconsistent, and because of methodological discrepancies, the findings remain controversial. Alcohol-feeding studies in AD mice support the notion that high alcohol intake promotes AD, while also hinting that low alcohol doses may be protective against AD. Chronic alcohol feeding to AD mice that delivers alcohol doses sufficient to cause liver injury largely promotes and accelerates AD pathology. The mechanisms by which alcohol can modulate cerebral AD pathology include Toll-like receptors, protein kinase-B (Akt)/mammalian target of rapamycin (mTOR) pathway, cyclic adenosine monophosphate (cAMP) response element-binding protein phosphorylation pathway, glycogen synthase kinase 3-β, cyclin-dependent kinase-5, insulin-like growth factor type-1 receptor, modulation of β-amyloid (Aβ) synthesis and clearance, microglial mediated, and brain endothelial alterations. Besides these brain-centric pathways, alcohol-mediated liver injury may significantly affect brain Aβ levels through alterations in the peripheral-to-central Aβ homeostasis. This article reviews published experimental studies (cell culture and AD rodent models) to summarize the scientific evidence and probable mechanisms (both cerebral and hepatic) by which alcohol promotes or protects against AD progression.

HIPK1 Inhibition Protects against Pathological Cardiac Hypertrophy by Inhibiting the CREB-C/EBPβ Axis.

Inhibition of pathological cardiac hypertrophy is recognized as an important therapeutic strategy for heart failure, although effective targets are still lacking in clinical practice. Homeodomain interacting protein kinase 1 (HIPK1) is a conserved serine/threonine kinase that can respond to different stress signals, however, whether and how HIPK1 regulates myocardial function is not reported. Here, it is observed that HIPK1 is increased during pathological cardiac hypertrophy. Both genetic ablation and gene therapy targeting HIPK1 are protective against pathological hypertrophy and heart failure in vivo. Hypertrophic stress-induced HIPK1 is present in the nucleus of cardiomyocytes, while HIPK1 inhibition prevents phenylephrine-induced cardiomyocyte hypertrophy through inhibiting cAMP-response element binding protein (CREB) phosphorylation at Ser271 and inactivating CCAAT/enhancer-binding protein β (C/EBPβ)-mediated transcription of pathological response genes. Inhibition of HIPK1 and CREB forms a synergistic pathway in preventing pathological cardiac hypertrophy. In conclusion, HIPK1 inhibition may serve as a promising novel therapeutic strategy to attenuate pathological cardiac hypertrophy and heart failure.

Endothelial depletion of Atg7 triggers astrocyte-microvascular disassociation at blood-brain barrier.

Microvascular basement membrane (BM) plays a pivotal role in the interactions of astrocyte with endothelium to maintain the blood-brain barrier (BBB) homeostasis; however, the significance and precise regulation of the endothelial cell-derived BM component in the BBB remain incompletely understood. Here, we report that conditional knockout of Atg7 in endothelial cells (Atg7-ECKO) leads to astrocyte-microvascular disassociation in the brain. Our results reveal astrocytic endfeet detachment from microvessels and BBB leakage in Atg7-ECKO mice. Furthermore, we find that the absence of endothelial Atg7 downregulates the expression of fibronectin, a major BM component of the BBB, causing significantly reduced coverage of astrocytes along cerebral microvessels. We reveal Atg7 triggers the expression of endothelial fibronectin via regulating PKA activity to affect the phosphorylation of cAMP-responsive element-binding protein. These results suggest that Atg7-regulated endothelial fibronectin production is required for astrocytes adhesion to microvascular wall for maintaining the BBB homeostasis. Thus, endothelial Atg7 plays an essential role in astrocyte-endothelium interactions to maintain the BBB integrity.

Apigenin Attenuates Hippocampal Microglial Activation and Restores Cognitive Function in Methotrexate-Treated Rats: Targeting the miR-15a/ROCK-1/ERK1/2 Pathway

Microglial activation underpins the methotrexate (MTX)-induced neurotoxicity; however, the precise mechanism remains unclear. This study appraised the potential impact of apigenin (Api), a neuroprotective flavonoid, in MTX-induced neurotoxicity in rats in terms of microglial activation through targeting the miR-15a/Rho-associated protein kinase-1 (ROCK-1)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Male Sprague Dawley rats were randomly divided into 4 groups: Normal control (saline i.p. daily and i.v. on days 8 and 15); Api control (20 mg/kg, p.o.) daily for 30 days; MTX-alone (75 mg/kg, i.v.) on days 8 and 15, then four i.p. injections of leucovorin (LCV): 6 mg/kg after 18 h, then three doses (3 mg/kg) every 8 h post-MTX; and Api co-treated (20 mg/kg/day, p.o.) throughout the model for 30 days, with administration of MTX and LCV as in group 3. MTX administration elevated hippocampal ionized calcium-binding adaptor protein-1 (Iba-1) immunostaining, indicating microglial activation. This was accompanied by neuroinflammation, oxidative stress, and enhanced apoptosis manifested by elevated hippocampal interleukin-1β, malondialdehyde, and caspase-3, and decreased reduced glutathione levels. Concurrently, abated miR-15a expression, overexpression of its target ROCK-1, diminished downstream ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation, and decreased hippocampal brain-derived neurotrophic factor (BDNF) levels were observed. Api mitigated the MTX-induced neurotoxicity by reversing the biochemical, histopathological, and behavioral derangements tested by novel object recognition and Morris water maze tests. Conclusively, Api lessens MTX-induced neuroinflammation, oxidative stress, and apoptosis and boosts cognitive function through inhibiting microglial activation via modulating the miR-15a/ROCK-1/ERK1/2/CREB/BDNF pathway.Graphical Graphical abstract showing the effects of methotrexate and apigenin co-treatment in MTX-induced neurotoxicity model.On the left, methotrexate (MTX) administration to rats resulted in hippocampal miR-15a downregulation, which triggered an enhanced expression of its target ROCK-1, consequently inhibiting the downstream ERK1/2/CREB/BDNF pathway, instigating a state of microglial activation, neuroinflammation, oxidative stress, and apoptosis. On the other hand, apigenin (Api) co-treatment restored miR-15a, inhibited ROCK-1 expression, and activated the ERK1/2/CREB/BDNF pathway, leading to diminished hippocampal microglial activation, neuroinflammation, and apoptosis, and restoration of the redox balance, along with improvement in memory and cognitive function of the MTX-treated rats.

Adrenergic receptor signaling regulates the CD40-receptor mediated anti-tumor immunity.

Anti-CD40 agonistic antibody (αCD40), an activator of dendritic cells (DC) can enhance antigen presentation and activate cytotoxic T-cells against poorly immunogenic tumors. However, cancer immunotherapy trials also suggest that αCD40 is only moderately effective in patients, falling short of achieving clinical success. Identifying factors that decrease αCD40 immune-stimulating effects can aid the translation of this agent to clinical reality. Here, we reveal that β-adrenergic signaling on DCs directly interferes with αCD40 efficacy in immunologically cold head and neck tumor model. We discovered that β-2 adrenergic receptor (β2AR) activation rewires CD40 signaling in DCs by directly inhibiting the phosphorylation of IκBα and indirectly by upregulating levels of phosphorylated-cAMP response element-binding protein (pCREB). Importantly, the addition of propranolol, a pan β-Blocker reprograms the CD40 pathways, inducing superior tumor regressions, increased infiltration of cytotoxic T-cells, and a reduced burden of regulatory T-cells in tumors compared to monotherapy. Thus, our study highlights an important mechanistic link between stress-induced β2AR signaling and reduced αCD40 efficacy in cold tumors, providing a new combinatorial approach to improve clinical outcomes in patients.