Inhibition of ZDHHC16 promoted osteogenic differentiation and reduced ferroptosis of dental pulp stem cells by CREB.

BACKGROUND: The repair of bone defects caused by periodontal diseases is a difficult challenge in clinical treatment. Dental pulp stem cells (DPSCs) are widely studied for alveolar bone repair. The current investigation aimed to examine the specific mechanisms underlying the role of Zinc finger DHHC-type palmitoyl transferases 16 (ZDHHC16) in the process of osteogenic differentiation (OD) of DPSCs. METHODS: The lentiviral vectors ZDHHC16 or si-ZDHHC16 were introduced in the DPSCs and then the cells were induced by an odontogenic medium for 21 days. Subsequently, Quantitate Polymerase Chain Reaction (PCR), immunofluorescent staining, proliferation assay, ethynyl deoxyuridine (EdU) staining, and western blot analysis were used to investigate the specific details of ZDHHC16 contribution in OD of DPSCs. RESULTS: Our findings indicate that ZDHHC16 exhibited a suppressive effect on cellular proliferation and oxidative phosphorylation, while concurrently inducing ferroptosis in DPSCs. Moreover, the inhibition of ZDHHC16 promoted cell development and OD and reduced ferroptosis of DPSCs. The expression of p-CREB was suppressed by ZDHHC16, and immunoprecipitation (IP) analysis revealed that ZDHHC16 protein exhibited interconnection with cAMP-response element binding protein (CREB) of DPSCs. The CREB suppression reduced the impacts of ZDHHC16 on OD and ferroptosis of DPSCs. The activation of CREB also reduced the influences of si-ZDHHC16 on OD and ferroptosis of DPSCs. CONCLUSIONS: These findings provide evidences to support a negative association between ZDHHC16 and OD of DPSCs, which might be mediated by ferroptosis of DPSCs via CREB.

Effects of exogenous energy on synthesis of steroid hormones and expression characteristics of the CREB/StAR signaling pathway in theca cells of laying hen.

Energy and the cAMP-response element binding protein (CREB)/steroidogenic acute regulatory protein (StAR) signaling pathway play important roles in steroid hormone production and follicular development in hens. This present study aimed to investigate the effects of exogenous energy on the synthesis of steroid hormones and the expression characteristics of the CREB/StAR signaling pathway in theca cells of laying hen. The primary theca cells of small yellow follicles were randomly divided into 6 treatments and cultured in medium with glucose concentrations of 1, 1.5, 3, 4.5, 6, and 7.5 mg/mL for 48 h. It was found that growth was robust and cell outlines were clear when cells were cultured with 1, 1.5, 3, and 4.5 mg/mL glucose, but cell viability was diminished and cell density decreased after exposure to glucose at 6 and 7.5 mg/mL for 48 h. Cell viability showed an increasing and then decreasing quadratic response to increasing glucose concentration in culture (r2 = 0.688, P < 0.001). The cell viability of theca cells cultured with 4.5 mg/mL glucose was greater than those cultured with 1, 1.5, 6, and 7.5 mg/mL glucose (P < 0.05). The concentration of estradiol in the medium containing 3 mg/mL glucose was higher than in medium containing 1, 1.5, and 6 mg/mL glucose (P < 0.05). There was an increasing and then decreasing quadratic correlation between progesterone concentrations and glucose concentrations (r2 = 0.522, P = 0.002). The concentration of progesterone in medium with 4.5 mg/mL glucose was higher than in medium with 1 and 7.5 mg/mL glucose (P < 0.05). There was an increasing and then decreasing quadratic correlation between the relative expression of CREB1 (r2 = 0.752, P < 0.001), StAR (r2 = 0.456, P = 0.002), CYP1B1 (r2 = 0.568, P < 0.001), and 3ß-HSD (r2 = 0.319, P = 0.018) in theca cells of laying hens and glucose concentrations after treatment with different glucose concentrations for 48 h. After treatment with 4.5 mg/mL glucose, the expression of StAR, CYP1B1, and 3ß-HSD genes were increased compared to treatment with 1, 1.5, 3, 6, and 7.5 mg/mL glucose (P < 0.001). There was an increasing and then decreasing quadratic correlation between glucose concentrations and protein expression of CREB1 (r2 = 0.819, P < 0.001), StAR (r2 = 0.844, P < 0.001), 3ß-HSD (r2 = 0.801, P < 0.001), and CYP11A1 (r2 = 0.800, P < 0.001) in theca cells of laying hens. The protein expression of CREB1, StAR, and 3ß-HSD in theca cells cultured with 4.5 mg/mL glucose was higher than in other groups (P < 0.001). The results indicate that the appropriate glucose concentration (4.5 mg/mL) can improve the synthesis of steroid hormones in theca cells of laying hens through the upregulation of key genes and proteins in the CREB/StAR signaling pathway.

Ceramide enhanced the hepatic glucagon response through regulation of CREB activity.

BACKGROUND & AIMS: Hyperglycemia is associated with lipid disorders in patients with diabetes. Ceramides are metabolites involved in sphingolipid metabolism that accumulate during lipid disorders and exert deleterious effects on glucose and lipid metabolism. However, the effects of ceramide on glucagon-mediated hepatic gluconeogenesis remain largely unknown. This study was designed to investigate the impact of ceramides on gluconeogenesis in the context of the hepatic glucagon response, with the aim of finding new pharmacological interventions for hyperglycemia in diabetes. METHODS: Liquid chromatography-mass spectrometry was used to quantify ceramide content in the serum of patients with diabetes. Primary hepatocytes were isolated from male C57BL/6J mice to study the effects of ceramide on hepatic glucose production. Immunofluorescence staining was performed to view cAMP-responsive element-binding protein (CREB)- regulated transcription co-activator 2 (CRTC2) nuclear translocation in hepatocytes. Serine palmitoyl-transferase, long chain base subunit 2 (Sptlc2) knockdown mice were generated using an adeno-associated virus containing shRNA, and hepatic glucose production was assessed glucagon tolerance and pyruvate tolerance tests in mice fed a normal chow diet and high-fat diet. RESULTS: Increased ceramide levels were observed in the serum of patients newly diagnosed with type 2 diabetes. De novo ceramide synthesis was activated in mice with metabolic disorders. Ceramide enhanced hepatic glucose production in primary hepatocytes. In contrast, genetic silencing of Sptlc2 prevented this process. Mechanistically, ceramides de-phosphorylate CRTC2 (Ser 171) and facilitate its translocation into the nucleus for CREB activation, thereby augmenting the hepatic glucagon response. Hepatic Sptlc2 silencing blocked ceramide generation in the liver and thus restrained the hepatic glucagon response in mice fed a normal chow diet and high-fat diet. CONCLUSIONS: These data indicate that ceramide serves as an intracellular messenger that augments hepatic glucose production by regulating CRTC2/CREB activity in the context of the hepatic glucagon response, suggesting that CRTC2 phosphorylation might be a potential node for pharmacological interventions to restrain the hyperglycemic response during fasting in diabetes.

Protection of p-Coumaric acid against chronic stress-induced neurobehavioral deficits in mice via activating the PKA-CREB-BDNF pathway.

There is a body of evidence to suggest that chronic stress modulates neurochemical homeostasis, alters neuronal structure, inhibits neurogenesis and contributes to development of mental disorders. Chronic stress-associated mental disorders present common symptoms of cognitive impairment and depression with complex disease mechanisms. P-coumaric acid (p-CA), a natural phenolic compound, is widely distributed in vegetables, cereals and fruits. p-CA exhibits a wide range of health-related effects, including anti-oxidative-stress, anti-mutagenesis, anti-inflammation and anti-cancer activities. The current study aims to evaluate the therapeutic potential of p-CA against stress-associated mental disorders. We assessed the effect of p-CA on cognitive deficits and depression-like behavior in mice exposed to chronic restraint stress (CRS); we used network pharmacology, biochemical and molecular biological approaches to elucidate the underlying molecular mechanisms. CRS exposure caused memory impairments and depression-like behavior in mice; p-CA administration attenuated these CRS-induced memory deficits and depression-like behavior. Network pharmacology analysis demonstrated that p-CA was possibly involved in multiple targets and a variety of signaling pathways. Among them, the protein kinase A (PKA) - cAMP-response element binding protein (CREB) - brain derived neurotrophic factor (BDNF) signaling pathway was predominant and further characterized. The levels of PKA, phosphorylated CREB (pCREB) and BDNF were significantly lowered in the hippocampus of CRS mice, suggesting disruption of the PKA-CREB-BDNF signaling pathway; p-CA treatment restored the signaling pathway. Furthermore, CRS upregulated expression of proinflammatory cytokines in hippocampus, while p-CA reversed the CRS-induced effects. Our findings suggest that p-CA will offer therapeutic benefit to patients with stress-associated mental disorders.

Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

Neuromodulatory effect of 4-(methylthio)butyl isothiocyanate against 3-nitropropionic acid induced oxidative impairments in human dopaminergic SH-SY5Y cells via BDNF/CREB/TrkB pathway.

Mitochondrial impairment, energetic crisis and elevated oxidative stress have been demonstrated to play a pivotal role in the pathological processes of Huntington's disease (HD). 3-Nitropropionic acid (3-NPA) is a natural neurotoxin that mimics the neurological dysfunctions, mitochondrial impairments and oxidative imbalance of HD. The current investigation was undertaken to demonstrate the neuroprotective effect of 4-(methylthio)butyl isothiocyanate (4-MTBITC) against the 3-NPA induced neurotoxicity in human dopaminergic SH-SY5Y cells. The experimental evidence of oxidative DNA damage by 3-NPA was elucidated by pBR322 DNA nicking assay. In contrast, the 4-MTBITC considerably attenuated the DNA damage, suggesting its free radical scavenging action against 3-NPA and Fenton's reagent. The dose and time-dependent increase of 3-NPA revealed its neurotoxic dose as 0.5 mM after 24 h of treatment of SH-SY5Y cells in MTT assay. In order to determine the optimal dose at which 4-MTBITC protects cell death, the 3-NPA (IC50) induced cells were pretreated with different concentrations of 4-MTBITC for 1 h. The neuroprotective dose of 4-MTBITC against 3-NPA was found to be 0.25 µM. Additionally, the elevated GSH levels in cells treated with 4-MTBITC indicate its propensity to eliminate reactive species generated as a result of 3-NPA-induced mitochondrial dysfunction. Likewise, it was determined through microscopic and flow cytometric experiments that 3-NPA's induced overproduction of reactive species and a decline in mitochondrial membrane potential (MMP) could be efficiently prevented by pre-treating cells with 4-MTBITC. To elucidate the underlying molecular mechanism, the RT-qPCR analysis revealed that the pre-treatment of 4-MTBITC effectively protected neuronal cells against 3-NPA-induced cell death by preventing Caspase-3 activation, Brain-derived neurotrophic factor (BDNF) upregulation, activation of cAMP response element-binding protein (CREB) and Nrf2 induction. Together, our findings lend credence to the idea that pre-treatment with 4-MTBITC reduced 3-NPA-induced neurotoxicity by lowering redox impairment, apoptotic state, and mitochondrial dysfunction. The present work, in conclusion, presented the first proof that the phytoconstituent 4-MTBITC supports the antioxidant system, BDNF/TrkB/CREB signaling, and neuronal survival in dopaminergic SH-SY5Y cells against 3-NPA-induced oxidative deficits.

Dexmedetomidine attenuates myocardial ischemia-reperfusion injury via inhibiting ferroptosis by the cAMP/PKA/CREB pathway.

This study is to investigate the effects of dexmedetomidine on myocardial ischemia-reperfusion (I/R) injury and its molecular mechanisms. H9c2 cell injury model was constructed by the hypoxia/normoxia (H/R) conditions. Besides, cAMP response element-binding protein (CREB) overexpression and knockdown cell lines were constructed. Cell viability was determined by cell-counting kit 8. Biochemical assays were used to detect oxidative stress-related biomarkers, cell apoptosis, and ferroptosis-related markers. Our results showed that dexmedetomidine's protective effects on H/R-induced cell damage were reversed by the inhibition of protein kinase A (PKA), CREB, and extracellular signal regulated kinase 1/2 (ERK1/2). Treatment of dexmedetomidine ameliorated oxidative stress in the cardiomyocytes induced by H/R, whereas inhibition of PKA, CREB, or ERK1/2 reversed these protective effects. Cell death including cell necrosis, apoptosis, and ferroptosis was found in the cells under H/R insult. Interestingly, targeting CREB ameliorated ferroptosis and oxidative stress in these cells. In conclusion, dexmedetomidine attenuates myocardial I/R injury by suppressing ferroptosis through the cAMP/PKA/CREB signaling pathway.

MicroRNA-582-5p targeting Creb1 modulates apoptosis in cardiomyocytes hypoxia/reperfusion-induced injury.

BACKGROUND: Myocardial ischemia-reperfusion injury (MIRI) caused by the reperfusion therapy of myocardial ischemic diseases is a kind of major disease that threatens human health and lives severely. There are lacking of effective therapeutic measures for MIRI. MicroRNAs (miRNAs) are abundant in mammalian species and play a critical role in the initiation, promotion, and progression of MIRI. However, the biological role and molecular mechanism of miRNAs in MIRI are not entirely clear. METHODS: We used bioinformatics analysis to uncover the significantly different miRNA by analyzing transcriptome sequencing data from myocardial tissue in the mouse MIRI model. Multiple miRNA-related databases, including miRdb, PicTar, and TargetScan were used to forecast the downstream target genes of the differentially expressed miRNA. Then, the experimental models, including male C57BL/6J mice and HL-1 cell line, were used for subsequent experiments including quantitative real-time polymerase chain reaction analysis, western blot analysis, hematoxylin and eosin staining, flow cytometry, luciferase assay, gene interference, and overexpression. RESULTS: MiR-582-5p was found to be differentially upregulated from the transcriptome sequencing data. The elevated levels of miR-582-5p were verified in MIRI mice and hypoxia/reperfusion (H/R)-induced HL-1 cells. Functional experiments revealed that miR-582-5p promoted apoptosis of H/R-induced HL-1 cells via downregulating cAMP-response element-binding protein 1 (Creb1). The inhibiting action of miR-582-5p inhibitor on H/R-induced apoptosis was partially reversed after Creb1 interference. CONCLUSIONS: Collectively, the research findings reported that upregulation of miR-582-5p promoted H/R-induced cardiomyocyte apoptosis by inhibiting Creb1. The potential diagnostic and therapeutic strategies targeting miR-582-5p and Creb1 could be beneficial for the MIRI treatment.

Oxytocin treatment improves dexamethasone-induced depression-like symptoms associated with enhancement of hippocampal CREB-BDNF signaling in female mice.

AIMS: Chronic stress and glucocorticoid exposure are risk factors for depression. Oxytocin (OT) has been shown to have antistress and antidepressant-like effects in male rodents. However, depression is twice as common in women than in men, and it remains unclear whether OT exerts antidepressant-like effects in women with depression. Therefore, in this study, we investigated the therapeutic effect of chronic OT administration in a female mouse model of dexamethasone (DEX)-induced depression. METHODS: Female C57BL/6J mice were administered saline (vehicle, s.c.), DEX (s.c.), or OT (i.p.) + DEX (s.c.) daily for 8 weeks, and then assessed for anxiety- and depression-like behaviors. We also examined the hippocampal levels of phosphorylated cAMP response element-binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF), which are important mediators of the response to antidepressants. RESULTS: Simultaneous OT treatment blocked the adverse effects of DEX on emotional behaviors. Furthermore, it upregulated p-CREB and BDNF in the hippocampus. CONCLUSION: OT may exert antidepressant-like effects by activating hippocampal CREB-BDNF signaling in a female mouse model of depression.

Gao-Zi-Yao improves learning and memory function in old spontaneous hypertensive rats.

AIMS: Gao-Zi-Yao has long been a unique way for treating various diseases. The present study is to explore the effect of Gao-Zi-Yao on learning and memory function in old spontaneous hypertensive rats (SHR) and its possible mechanism. METHOD: Male old SHR were received different doses of Gao-Zi-Yao for 4 weeks. Systolic blood pressure (SBP) and heart rate were monitored. Serum levels of nitric oxide (NO), interleukin (IL)-1ß, IL-2, and tumor necrotic factor (TNF)-α were measured. Morris water maze was performed to test the learning and memory function of the rats. Number of neurons in hippocampus was counted by Nissl staining. Western blot was applied to detect the expressions of learning and memory function related proteins, N-methyl-d-aspartate receptor 2B (NMDAR 2B), glutamate receptor 1 (GluR1), phosphorylated-calmodulin-dependent protein kinase II (p-CaMK II), and phosphorylated-cAMP responsive element-binding protein (p-CREB) in rat hippocampus. RESULTS: Data showed that Gao-Zi-Yao reduced SBP in old SHR, elevated NO level, and suppressed levels of IL-1ß, IL-2, TNF-α. The results of Morris water maze experiment showed that Gao-Zi-Yao dose-dependently improved learning and memory function. Number of neurons in the hippocampal dentate gyrus (DG) region of the old SHR was increased by Gao-Zi-Yao treatment. In addition, Gao-Zi-Yao elevated the protein expressions of NMDAR 2B, GluR1, p-CaMK II, and p-CREB in hippocampus. CONCLUSION: Gao-Zi-Yao decreases SBP and improves the learning and memory function of the old SHR by regulation of oxidative stress, inflammatory factors and neuron number in hippocampal DG area and the expression of learning and memory function related proteins.

[Effect of miR-203/CREB1 Signaling Regulation Mediated by DNA Methylation on the Proliferation and Apoptosis of Multiple Myeloma Cells].

OBJECTIVE: To investigate the effect of miR-203/CREB1 signaling regulation mediated by DNA methylation on the proliferation, invasion and apoptosis of multiple myeloma (MM) cells. METHODS: The methylation level of miR-203 in the RPMI 8226 cells was detected by bisulfite sequcucing polymerase chain reaction (BSP). The mRNA expression of miR-203 was measured by quantitative real-time polymerase chain reaction. RPMI 8226 cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). The miR-203 mimic in MM cell line RPMI 8226 was transfected to establish overexpressed miR-203 cell. The proliferation, invasion ability and apoptosis of RPMI 8226 cell was detected by CCK-8 assay, Transwell, and flow cytometry, respectively. The targeting relationship between miR-203 and CREB1 was verified by double luciferase report assay. Western blot was used to detect the expression of CREB1 protein. RESULTS: Hypermethylation of miR-203 promoter region and low expression level of miR-203 mRNA were detected in the RPMI 8226 cells, which showed that demethylation could induce the expression of miR-203. The proliferation and invasion ability of RPMI 8226 cells after treated by 5-Aza-CdR were inhibited, and showed statistical significance as compared with blank control group (both P<0.05)，while the apoptosis rate was promoted (P<0.05). The proliferation, invasion ability and apoptosis of overexpressed miR-203 were the same as the demethylation group. Double luciferase report assay confirmed that CREB1 was the direct target of miR-203. The protein level of CREB1 was inhibited by demethylation and showed statistical significance as compared with control group (P<0.05). CONCLUSION: MiR-203 targeting CREB1 mediated by DNA methylation leads to maintain the malignant biological behaviors of MM cells.

Silibinin promotes melanogenesis through the PKA and p38 MAPK signaling pathways in melanoma cells.

Silibinin is a flavonolignan isolated from milk thistle (Silybum marianum). Silibinin has been reported to possess multiple biological activities; however, its effect on melanogenesis remains unclear. This study investigated the effect of silibinin on melanogenesis in melanoma cells and the associated molecular mechanism. Our findings demonstrated that silibinin markedly increased melanin content in murine B16-F1 and human HMV-II melanoma cells. Silibinin activated intracellular tyrosinase activity and expression of tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, and microphthalmia-associated transcription factor (MITF). Furthermore, silibinin enhanced the phosphorylation of cyclic AMP-responsive element-binding protein (CREB), protein kinase A (PKA), and p38 mitogen-activated protein kinase (MAPK) but not of Akt and extracellular signal-regulated kinase (ERK). The specific PKA (H-89) and p38 (SB203580) inhibitors significantly attenuated silibinin-mediated melanin synthesis. These results suggest that silibinin is an effective stimulator of melanogenesis through upregulation of the protein expression of melanogenic enzymes activated by the PKA and p38 pathways, leading to CREB phosphorylation and MITF expression. Therefore, silibinin may have potential for use in the treatment of hypopigmentation disorders.

Purkinje cell-specific deletion of CREB worsens alcohol-induced cerebellar neuronal losses and motor deficits.

INTRODUCTION: Exposure to alcohol during pregnancy can kill developing fetal neurons and lead to fetal alcohol spectrum disorder (FASD) in the offspring. However, not all fetuses are equally vulnerable to alcohol toxicity. These differences in vulnerability among individuals are likely due, at least in part, to genetic differences. Some genes encode neuroprotective molecules that act through signaling pathways to protect neurons against alcohol's toxic effects. One signaling pathway that can protect cultured neurons against alcohol-induced cell death in vitro is the cAMP pathway. A goal of this study was to determine whether the cAMP pathway can exert a similar neuroprotective effect against alcohol in vivo. A key molecule within the cAMP pathway is cAMP response element binding protein (CREB). In this study, CREB was specifically disrupted in cerebellar Purkinje cells to study its role in protection of cerebellar neurons against alcohol toxicity. METHODS: Mice with Purkinje cell-specific knockout of CREB were generated with the Cre-lox system. A 2 × 2 design was used in which Cre-negative and Cre-positive mice received either 0.0 or 2.2 mg/g ethanol by intraperitoneal (i.p.) injection daily over postnatal day (PD) 4-9. Stereological cell counts of cerebellar Purkinje cells and granule cells were performed on PD 10. Motor function was assessed on PD 40 using the rotarod. RESULTS: Purkinje cell-specific disruption of CREB alone (in the absence of alcohol) induced only a small reduction in Purkinje cell number. However, the loss of CREB function from Purkinje cells greatly increased the vulnerability of Purkinje cells to alcohol-induced cell death. While alcohol killed 20% of Purkinje cells in the Cre-negative (CREB-expressing) mice, alcohol killed 57% of Purkinje cells in the Cre-positive (CREB-nonexpressing) mice. This large loss of Purkinje cells did not lead to similar alcohol-induced losses of granule cells. In the absence of alcohol, lack of CREB function in Purkinje cells had no effect on rotarod performance. However, in the presence of alcohol, disruption of CREB in Purkinje cells substantially worsened rotarod performance. DISCUSSION: Disruption of a single gene (CREB) in a single neuronal population (Purkinje cells) greatly increases the vulnerability of that cell population to alcohol-induced cell death and worsens alcohol-induced brain dysfunction. The results suggest that the cAMP pathway can protect cells in vivo against alcohol toxicity and underline the importance of genetics in determining the neuropathology and behavioral deficits of FASD.

Antidepressant-like effect of male mating behavior through oxytocin-induced CREB signaling.

Male sexual activity reduces the level of depression through oxytocin (OT) release within the brain. In this study, we showed that male mating behavior reduces depression-like behavior through OT-induced cAMP response element binding protein (CREB) signaling in the hippocampus. Moreover, we showed that mating behavior in wild-type (WT) male mice increased CREB phosphorylation in hippocampus, whereas that OT receptor knockout (OTR KO) male mice had no effect on CREB phosphorylation. CREB phosphorylation in hippocampus was also increased after OT induction in hippocampal slices prepared from WT mice. In addition, male mating behavior induced the expression of brain-derived neurotrophic factor (BDNF), which was not observed in OTR KO mice. Antidepressant-like effect of mating behavior had no effect in OTR KO mice. These findings suggest that male sexual activity has antidepressant effects through OT-induced CREB signaling in the hippocampus.

Antidepressant effects of Enterococcus faecalis 2001 through the regulation of prefrontal cortical myelination via the enhancement of CREB/BDNF and NF-κB p65/LIF/STAT3 pathways in olfactory bulbectomized mice.

A therapeutic strategy through the gut-brain axis has been proven to be effective in treatment for depression. In our previous study, we demonstrated that Enterococcus faecalis 2001 (EF-2001) prevents colitis-induced depressive-like behavior through the gut-brain axis in mice. More recently, we found that demyelination in the prefrontal cortex (PFC) was associated with depressive-like behavior in an animal model of major depressive disorder, olfactory bulbectomized (OBX) mice. The present study investigated the effects of EF-2001 on depressive-like behaviors in OBX mice and the underlying molecular mechanisms from the perspective of myelination in the PFC. OBX mice exhibited depressive-like behaviors in the tail-suspension, splash, and sucrose preference tests, and decreased myelin and paranodal proteins along with mature oligodendrocytes in the PFC. These behavioral and biochemical changes were all prevented by treatment with EF-2001. Further, EF-2001 treatment increased brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) in the PFC. Interestingly, an immunohistochemical analysis revealed enhanced phospho (p) -cAMP-responsive element binding protein (CREB) expression in neurons, p-nuclear factor-kappa B (NFκB) p65 (Ser536) expression in astrocytes, and p-signal transducer and activator of transcription 3 (STAT3) (Ty705) expression in mature oligodendrocytes in the PFC of OBX mice. From these results, we suggest that EF-2001 administration prevents depressive-like behaviors by regulating prefrontal cortical myelination via the enhancement of CREB/BDNF and NFκB p65/LIF/STAT3 pathways. Our findings strongly support the idea that a therapeutic strategy involving the gut microbiota may be a promising alternative treatment for alleviating symptoms of depression.

Adipose expression of CREB3L3 modulates body weight during obesity.

We found the hepatic transcription factor Cyclic-AMP Responsive Element Binding Protein 3-like-3 (CREB3L3) to be expressed in adipose tissue, and selectively downregulated in the more metabolically protective subcutaneous adipose tissue in obese mice and humans. We sought to elucidate the specific role of this factor in adipose biology. CREB3L3 fat-specific knockout mice were fed a high-fat diet to induce obesity and metabolic dysfunction. Additionally, we injected a flip-excision adeno-associated virus directly into the subcutaneous inguinal adipose tissue of Adiponectin-Cre mice to create a depot-specific overexpression model for further assessment. Fat-specific ablation of CREB3L3 enhanced weight gain and insulin resistance following high-fat feeding, as fat-specific knockout mice expended less energy and possessed more inflammatory adipose tissue. Conversely, inguinal fat CREB3L3 overexpression deterred diet-induced obesity and ameliorated metabolic dysfunction. Together, this study highlights the relevance of CREB3L3 in obese adipose tissue and demonstrates its role as a powerful body weight modulator.

Impairments of exploration and memory after systemic or prelimbic D1-receptor antagonism in rats.

D1-receptor antagonism is known to impair rodent memory but also inhibits spontaneous exploration of stimuli to be remembered. Hypo-exploration could contribute to impaired memory by influencing event processing. In order to explore this effect, the D1 receptor antagonist, SCH23390, was administered to rats via routes that either did or did not affect spontaneous exploration: systemic or prelimbic administration, respectively. Effects were tested in spatial and non-spatial memory tasks selected for their requirements for self-initiated exploration of stimuli to be remembered in order to examine the effects on memory: cross-maze and object recognition task. Systemic administration reduced spatial exploration in cross-maze as well as in an open field test, and also reduced object exploration. Spatial (hippocampus-dependent) short-term memory was inhibited in the cross-maze and non-spatial short-term object retention was also impaired. In contrast to these systemic effects, bilateral injections of SCH23390 into the prelimbic cortices altered neither spatial nor object exploration, but did inhibit short-term memory in both cross-maze and object recognition task. Therefore, the inhibiting effects of SCH23390 on both spatial and non-spatial memory were not mediated indirectly via reduced exploration of stimuli to be remembered, but through antagonism of a prelimbic D1-R function that is directly involved in memory formation. Finally, a cooperative regulation of spatial exploration between D1-R and mGlu5 was indicated by a synergistic effect of the antagonists SCH23390 and MPEP.

4-Methylnitrosamino-1-3-pyridyl-1-butanone (NNK) promotes lung cancer cell survival by stimulating thromboxane A2 and its receptor.

The role of thromboxane A(2) (TxA(2)) in smoking-associated lung cancer is poorly understood. This study was conducted to study the role of TxA(2) in smoking carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-promoted cell survival and growth in human lung cancer cells. We found that NNK increased TxA(2) synthase (TxAS) expression and thromboxane B(2) (TxB(2)) generation in cultured lung cancer cells, the result of which was supported by the increased level of TxAS in lung cancer tissues of smokers. Both TxAS-specific inhibitor furegrelate and TxA(2) receptor antagonist SQ29548 completely blocked NNK-mediated cell survival and growth via inducting apoptosis. TxA(2) receptor agonist U46619 reconstituted a near-full survival and growth response to NNK when TxAS was inhibited, affirming the role of TxA(2) receptor in NNK-mediated cell survival and growth. Suppression of cyclic adenosine monophosphate response element binding protein (CREB) activity by its small interference RNA blocked the effect of NNK. Phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) also had a positive role. Altogether, our results have revealed that NNK stimulates TxA(2) synthesis and activates its receptor in lung cancer cells. The increased TxA(2) may then activate CREB through PI3K/Akt and extracellular ERK pathways, thereby contributing to the NNK-promoted survival and growth of lung cancer cells.

NDRG2 gene expression in B16F10 melanoma cells restrains melanogenesis via inhibition of Mitf expression.

NDRG2 (N-myc downstream-regulated gene 2) is a candidate tumor suppressor implicated in control of glioblastoma proliferation and dendritic cell differentiation. The microphthalmia-associated transcription factor (Mitf) plays a crucial role in the melanocyte lineage and in melanoma by controlling survival, differentiation, cell cycle entry and exit, and melanoma metastasis. Identifying upstream regulators of Mitf expression, therefore, remains a key issue. In this study, we aimed to assess whether the candidate tumor suppressor NDRG2 can modulate Mitf expression. Here, we show that NDRG2 acts to prevent cAMP and beta-catenin-mediated activation of the Mitf promoter, thereby blocking melanogenesis via the downstream Mitf target genes Tyrosinase, Tyrp1 and Dct. The data suggest that NDRG2 impairs melanogenesis by interfering with both the TCF/beta-catenin and cAMP/CREB pathways that are known to stimulate Mitf expression in melanocytes and have major implications for the role of NDRG2 in pigmentation and melanoma progression. Taken together, the results not only identify NDRG2 as a novel regulator of pigmentation, but also potentially a key factor in regulating melanoma progression via Mitf.

Human LZIP induces monocyte CC chemokine receptor 2 expression leading to enhancement of monocyte chemoattractant protein 1/CCL2-induced cell migration.

Chemokines and chemokine receptors play a role in migration of circulating leukocytes to the region of inflammation. Human LZIP is an uncharacterized transcription factor and is known to participate in leukotactin (Lkn)-1/CCL15-induced cell migration. We investigated the role of human LZIP in expression of CC chemokine receptors (CCRs) and its involvement in monocyte migration. RNase protection analysis showed that LZIP increased mRNA expression of CCR2 and CCR1 in THP-1 cells. Surface expressions of both CCR2 and CCR1 were also increased by LZIP. Results from an electrophoretic mobility shift assay showed that LZIP binds to the C/EBP element in the CCR2 promoter. LZIP also enhanced the chemotactic activities of monocyte chemoattractant protein-1/CCL2 and Lkn-1. These results suggest that LZIP regulates expression of chemokine receptors that are involved in monocyte migration.