The PKA-CREB1 axis regulates coronavirus proliferation by viral helicase nsp13 association.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a worldwide threat in the past 3 years. Although it has been widely and intensively investigated, the mechanism underlying the coronavirus-host interaction requires further elucidation, which may contribute to the development of new antiviral strategies. Here, we demonstrated that the host cAMP-responsive element-binding protein (CREB1) interacts with the non-structural protein 13 (nsp13) of SARS-CoV-2, a conserved helicase for coronavirus replication, both in cells and in lung tissues subjected to SARS-CoV-2 infection. The ATPase and helicase activity of viral nsp13 were shown to be potentiated by CREB1 association, as well as by Protein kinase A (PKA)-mediated CREB1 activation. SARS-CoV-2 replication is significantly suppressed by PKA Cα, cAMP-activated protein kinase catalytic subunit alpha (PRKACA), and CREB1 knockdown or inhibition. Consistently, the CREB1 inhibitor 666-15 has shown significant antiviral effects against both the WIV04 strain and the Omicron strain of the SARS-CoV-2. Our findings indicate that the PKA-CREB1 signaling axis may serve as a novel therapeutic target against coronavirus infection. IMPORTANCE: In this study, we provide solid evidence that host transcription factor cAMP-responsive element-binding protein (CREB1) interacts directly with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) helicase non-structural protein 13 (nsp13) and potentiate its ATPase and helicase activity. And by live SARS-CoV-2 virus infection, the inhibition of CREB1 dramatically impairs SARS-CoV-2 replication in vivo. Notably, the IC50 of CREB1 inhibitor 666-15 is comparable to that of remdesivir. These results may extend to all highly pathogenic coronaviruses due to the conserved nsp13 sequences in the virus.

Cyclic AMP-regulatory element-binding protein: a novel UV-targeted transcription factor in skin cancer.

Common therapeutics in relation to melanoma and non-melanoma cancers include the use of kinase inhibitors. The long-term benefits of kinases, however, are limited by development of drug resistance. An alternative approach for treatment would be to focus on transcription factors. Cyclic AMP-regulatory element-binding protein (CREB) is a transcription factor that is commonly overactivated or overexpressed in many different cancers including skin cancer. Ultraviolet radiation (UVR), one of the main causes of skin cancer, can activate CREB in both melanocytes and keratinocytes. In addition, CREB has been found to be activated in skin cancers. Considering the prominent role that CREB plays in skin cancers, the studies reviewed herein raise the possibility of CREB as a potential prognostic and diagnostic marker of skin cancer and a novel target for therapeutic intervention.

Integrative Cistromic and Transcriptomic Analyses Identify CREB Target Genes in Cystic Renal Epithelial Cells.

BACKGROUND: Genome-wide mapping of transcription factor (TF) binding sites is essential to identify a TF's direct target genes in kidney development and diseases. However, due to the cellular complexity of the kidney and limited numbers of a given cell type, it has been challenging to determine the binding sites of a TF in vivo. cAMP response element-binding protein (CREB) is phosphorylated and hyperactive in autosomal dominant polycystic kidney disease (ADPKD). We focus on CREB as an example to profile genomic loci bound by a TF and to identify its target genes using low numbers of specific kidney cells. METHODS: Cleavage under targets and release using nuclease (CUT&RUN) assays were performed with Dolichos biflorus agglutinin (DBA)-positive tubular epithelial cells from normal and ADPKD mouse kidneys. Pharmacologic inhibition of CREB with 666-15 and genetic inhibition with A-CREB were undertaken using ADPKD mouse models. RESULTS: CUT&RUN to profile genome-wide distribution of phosphorylated CREB (p-CREB) indicated correlation of p-CREB binding with active histone modifications (H3K4me3 and H3K27ac) in cystic epithelial cells. Integrative analysis with CUT&RUN and RNA-sequencing revealed CREB direct targets, including genes involved in ribosome biogenesis and protein synthesis. Pharmacologic and genetic inhibition of CREB suppressed cyst growth in ADPKD mouse models. CONCLUSIONS: CREB promotes cystogenesis by activating ribosome biogenesis genes. CUT&RUN, coupled with transcriptomic analysis, enables interrogation of TF binding and identification of direct TF targets from a low number of specific kidney cells.

Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP-Epac-CREB Signaling Pathway.

Fibroblast growth factor 21 (FGF21) functions as a polypeptide hormone to regulate glucose and lipid metabolism, and its expression is regulated by cellular metabolic stress. Pyruvate is an important intermediate metabolite that acts as a key hub for cellular fuel metabolism. However, the effect of pyruvate on hepatic FGF21 expression and secretion remains unknown. Herein, we examined the gene expression and protein levels of FGF21 in human hepatoma HepG2 cells and mouse AML12 hepatocytes in vitro, as well as in mice in vivo. In HepG2 and AML12 cells, pyruvate at concentrations above 0.1 mM significantly increased FGF21 expression and secretion. The increase in cellular cAMP levels by adenylyl cyclase activation, phosphodiesterase (PDE) inhibition and 8-Bromo-cAMP administration significantly restrained pyruvate-stimulated FGF21 expression. Pyruvate significantly increased PDE activities, reduced cAMP levels and decreased CREB phosphorylation. The inhibition of exchange protein directed activated by cAMP (Epac) and cAMP response element binding protein (CREB) upregulated FGF21 expression, upon which pyruvate no longer increased FGF21 expression. The increase in plasma pyruvate levels in mice induced by the intraperitoneal injection of pyruvate significantly increased FGF21 gene expression and PDE activity with a reduction in cAMP levels and CREB phosphorylation in the mouse liver compared with the control. In conclusion, pyruvate activates PDEs to reduce cAMP and then inhibits the cAMP-Epac-CREB signaling pathway to upregulate FGF21 expression in hepatocytes.

Hepatitis E viral infection regulates estrogen signaling pathways: Inhibition of the cAMPK-PKA-CREB and PI3K-AKT-mTOR signaling pathways.

Hepatitis E virus (HEV) infection has become a global concern with high mortality rates among pregnant women, especially those in their third trimester of pregnancy. Estrogen plays an important role in mediating the body, regulating physiological and pathological processes. Estrogen is activated by binding to estrogen receptors (ERs) and mediates rapid signaling events by pathways that involve transmembrane ERs. Our previous study had confirmed that high estrogen levels during pregnancy are associated with high HEV titers. However, the association between HEV infection and estrogen signaling pathways remains unclear. In the present study, the regulation of estrogen signaling pathways by HEV infection was evaluated. Results demonstrated that HEV infection significantly inhibits the cAMP-PKA-CREB and PI3K-AKT-mTOR signaling pathways, but is independent of the Ras-Raf-MEK-ERK signaling pathway. In summary, the increasing estrogen levels and highly activated ERα during pregnancy aggravates HEV replication. The exacerbation of HEV replication, in turn, inhibits ERα expression and suppresses both cAMP-PKA-CREB and PI3K-AKT-mTOR signaling pathways.

Diphlorethohydroxycarmalol inhibits melanogenesis via protein kinase A/cAMP response element-binding protein and extracellular signal-regulated kinase-mediated microphthalmia-associated transcription factor downregulation in α-melanocyte stimulating hormone-stimulated B16F10 melanoma cells and zebrafish.

Diphlorethohydroxycarmalol (DPHC) is a marine polyphenolic compound derived from brown alga Ishige okamurae. A previously study has suggested that DPHC possesses strong mushroom tyrosinase inhibitory activity. However, the anti-melanogenesis effect of DPHC has not been reported at cellular level. The objective of the present study was to clarify the melanogenesis inhibitory effect of DPHC and its molecular mechanisms in murine melanoma cells (B16F10) and zebrafish model. DPHC significantly inhibited tyrosinase activity and melanin content dose-dependently in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. This polyphenolic compound also suppressed the expression of phosphorylation of cAMP response element-binding protein (CREB) by attenuating phosphorylation of cAMP-dependent protein kinase A, resulting in decreased MITF expression levels. Furthermore, DPHC downregulated MITF protein expression levels by promoting the phosphorylation of extracellular signal-regulated kinase. It also inhibited tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2 in α-MSH stimulated B16F10 cells. In in vivo studies using zebrafish, DPHC also markedly inhibited melanin synthesis in a dose-dependent manner. These results demonstrate that DPHC can effectively inhibit melanogenesis in melanoma cells in vitro and in zebrafish in vivo, suggesting that DPHC could be applied in fields of pharmaceutical and cosmeceuticals as a skin-whitening agent. Significance of study: The present study showed for the first time that DPHC could inhibit a-MSH-stimulated melanogenesis via PKA/CREB and ERK pathway in melanoma cells. It also could inhibit pigmentation in vivo in a zebrafish model. This evidence suggests that DPHC has potential as a skin whitening agent. Taken together, DPHC could be considered as a novel anti-melanogenic agent to be applied in cosmetic, food, and medical industry.

Chronic administration of Tat-GluR23Y ameliorates cognitive dysfunction targeting CREB signaling in rats with amyloid beta neurotoxicity.

Alzheimer's disease (AD) is behaviorally characterized by memory impairments, and pathologically by amyloid ß1-42 (Aß1-42) plaques and tangles. Aß binds to excitatory synapses and disrupts their transmission due to dysregulation of the glutamate receptors. Here we hypothesized that chronic inhibition of the endocytosis of AMPA receptors together with GluN2B subunit of NMDA receptors might improve cognition deficit induced by Aß(1-42) neurotoxicity. Forty male Wistar rats were used in this study and divided into 5 groups: Saline + Saline, Aß+Saline, Aß+Ifen (Ifenprodil, 3 nmol /2 weeks), Aß+GluR23Y (Tat-GluR23Y 3 µmol/kg/2 weeks) and Aß+Ifen+GluR23Y (same doses and durations). Aß(1-42) neurotoxicity was induced by intracerebroventricular (ICV) injection of Aß1-42 (2 µg/µl/side), and then animals received the related treatments for 14 days. Cognitive performance of rats and hippocampal level of cAMP-response element-binding (CREB) were evaluated using Morris Water Maze (MWM), and western blotting respectively. Obtained data from the acquisition trials were analyzed by two way Anova and Student T test. Also one way Analysis of variance (ANOVA) with post hoc Tuckey were used to clarify between groups differences in probe test. The Group receiving Aß, showed significant cognition deficit (long latency to platform and short total time spent in target quadrant (TTS), parallel with lower level of hippocampal CREB, versus vehicle group. While, Aß+ GluR23Y exhibited the shortest latency to platform and the longest TTS during the probe test, parallel with the higher hippocampal level of CREB compared with other groups. The present study provides evidence that chronic administration of Tat-GluR23Y; an inhibitor of GluA2-AMPARs endocytosis, successfully restores spatial memory impaired by amyloid beta neurotoxicity targeting CREB signaling pathway.

The role of nucleus accumbens CREB attenuation in rescuing low voluntary running behavior in female rats.

Given the integral role of nucleus accumbens (NAc) cAMP response element binding protein (CREB) activity in motivational processes, the goal of the current study was to determine whether blunting chronic NAc CREB activity could rescue the low physical activity motivation of female, low voluntary running (LVR) rats. NAc CREB phosphorylation is elevated in these rats, a state previously attributed to deficits in reward valuation. It was recently shown that overexpression of the upstream CREB inhibitor, protein kinase inhibitor alpha (PKIα), increased LVR nightly running by ~threefold. Therefore, the current study addresses the extent to which NAc CREB attenuation influences female LVR and wild-type (WT) wheel-running behavior. Inducible reductions in NAc neuronal activity using Gi-coupled hM4Di DREADDs increased running behavior in LVR, but not in WT, rats. Similarly, site-directed pharmacological inhibition of NAc CREB activity significantly increased LVR nightly running distance and time by ~twofold, with no effect in WT rats. Finally, environmentally enriched LVR rats exhibit higher levels of running compared to socially isolated rats in what appeared to be a CREB-related manner. Considering the positive outcomes of upstream CREB modulation and environmental enrichment on LVR behavior, we believe that blunting NAc CREB activity has the neuromolecular potential to partially reverse low physical activity motivation, as exemplified by the LVR model. The positive physical activity outcome of early life enrichment adds translatable value to human childhood enrichment and highlights its importance on motivational processes later in life.

Aspects of cAMP Signaling in Epileptogenesis and Seizures and Its Potential as Drug Target.

Epilepsy is one of the most common chronic neurological conditions. Today, close to 30 different medications to prevent epileptic seizures are in use; yet, far from all patients become seizure free upon medical treatment. Thus, there is a need for new pharmacological approaches including novel drug targets for the management of epilepsy. Despite the fact that a role for cAMP signaling in epileptogenesis and seizures was first suggested some four decades ago, none of the current medications target the cAMP signaling system. The reasons for this are probably many including limited knowledge of the underlying biology and pathology as well as difficulties in designing selective drugs for the different components of the cAMP signaling system. This review explores selected aspects of cAMP signaling in the context of epileptogenesis and seizures including cAMP response element binding (CREB)-mediated transcriptional regulation. We discuss the therapeutic potential of targeting cAMP signaling in epilepsy and point to an increased knowledge of the A-kinase anchoring protein-based signaling hubs as being of seminal importance for future drug discovery within the field. Further, in terms of targeting CREB, we argue that targeting upstream cAMP signals might be more fruitful than targeting CREB itself. Finally, we point to astrocytes as cellular targets in epilepsy since cAMP signals may regulate astrocytic K+ clearance affecting neuronal excitability.

Mechanistic insights into the activation of ester prodrugs of 666-15.

cAMP-response element binding protein (CREB) is an oncogenic transcription factor implicated in many different types of cancer. We previously reported the discovery of 666-15 as a potent inhibitor of CREB-mediated gene transcription. In an effort to improve the aqueous solubility of 666-15, amino ester prodrugs 1 and 4 were designed and synthesized. Detailed chemical and biological studies of 1 and 4 revealed that a small portion of the prodrugs were converted into 666-15 through intermediate 3 instead of a long-range O,N-acyl transfer reaction that was initially proposed. These results provide unique insights into the activation of these ester prodrugs.

Small molecule nAS-E targeting cAMP response element binding protein (CREB) and CREB-binding protein interaction inhibits breast cancer bone metastasis.

Bone is the most common metastatic site for breast cancer. The excessive osteoclast activity in the metastatic bone lesions often produces osteolysis. The cyclic-AMP (cAMP)-response element binding protein (CREB) serves a variety of biological functions including the transformation and immortalization of breast cancer cells. In addition, evidence has shown that CREB plays a key role in osteoclastgenesis and bone resorption. Small organic molecules with good pharmacokinetic properties and specificity, targeting CREB-CBP (CREB-binding protein) interaction to inhibit CREB-mediated gene transcription have attracted more considerations as cancer therapeutics. We recently identified naphthol AS-E (nAS-E) as a cell-permeable inhibitor of CREB-mediated gene transcription through inhibiting CREB-CBP interaction. In this study, we tested the effect of nAS-E on breast cancer cell proliferation, survival, migration as well as osteoclast formation and bone resorption in vitro for the first time. Our results demonstrated that nAS-E inhibited breast cancer cell proliferation, migration, survival and suppressed osteoclast differentiation as well as bone resorption through inhibiting CREB-CBP interaction. In addition, the in vivo effect of nAS-E in protecting against breast cancer-induced osteolysis was evaluated. Our results indicated that nAS-E could reverse bone loss induced by MDA-MB-231 tumour. These results suggest that small molecules targeting CREB-CBP interaction to inhibit CREB-mediated gene transcription might be a potential approach for the treatment of breast cancer bone metastasis.

Discovery of selective inhibitors for cyclic AMP response element-binding protein: a combined ligand and structure-based resources pipeline.

Bromodomains are epigenetic readers of acetyl-lysine involved in chromatin remodeling and transcriptional regulations. Over the past few years, extensive research has been carried out to discover small-molecule inhibitors against bromodomains to treat various diseases. Cyclic AMP response element-binding protein (CREBBP) bromodomain has emerged as a hot target for cancer therapy. This study aims at discovering new inhibitors against CREBBP bromodomain using ligand-based molecular docking. A library of 2168 lead-like compounds were docked into the Kac binding site of CREBBP bromodomain. On the basis of the energy score and interaction analysis, six compounds were selected. In order to validate the stability of these six protein-ligand complexes 20 ns molecular dynamics simulations and principal component analyses were carried out. Based on the different analyses these six compounds may provide valuable information for developing CREBBP selective inhibitors.

CREB Protects against Temporal Lobe Epilepsy Associated with Cognitive Impairment by Controlling Oxidative Neuronal Damage.

BACKGROUND: Cognitive dysfunction as a common comorbidity of epilepsy often manifests as learning and memory impairments in patients with temporal lobe epilepsy (TLE). The pathogenetic molecular mechanisms underlying epilepsy-associated cognitive dysfunction are incompletely understood. We investigated the role of cAMP response element binding protein (CREB) and its downstream signaling pathways in the pathogenesis of cognitive impairment in mice with TLE. METHODS: Plasmid vectors of CREB-specific short-hairpin RNAs and CREB cDNA were prepared and transfected into primary neurons. Neuronal apoptosis and mitochondrial oxidative stress were assessed by flow cytometry. For in vivo studies, TLE in mice was induced by pilocarpine injection, and TLE-associated memory decline was evaluated using the Morris water maze after treatment with the CREB inhibitor 666-15, with or without the mitochondria-specific antioxidant MitoQ. CREB and its downstream mediators were examined by Western blotting analysis and quantitative reverse transcription polymerase chain reaction. RESULTS: CREB knockdown induced mitochondrial reactive oxygen species production and apoptosis in primary neurons whereas CREB overexpression brought the opposite effects. The TLE mice exhibited elevated oxidative stress and neuronal apoptosis with decreased expression of CREB and its downstream mediators including PKA, CaMKIV, arc, and c-fos. CREB inhibition exacerbated TLE-associated oxidative neuronal apoptosis and memory decline. MitoQ treatment restored the expression of CREB and its downstream mediators, and prevented TLE-associated oxidative neuronal damage and memory deficits aggravated by CREB inhibition. CONCLUSION: CREB plays a significant role in TLE-associated oxidative neuronal damage and memory impairment. This novel finding provides the evidence of the relationship between CREB and mitochondrial oxidative stress and cognitive dysfunction in epilepsy. Mitochondria-specific antioxidants such as MitoQ may alleviate TLE-associated cognitive dysfunction through activation of CREB and its downstream signaling pathways.

Calcium/Calmodulin-Dependent Protein Kinase IV Mediates IFN-γ-Induced Immune Behaviors in Skeletal Muscle Cells.

BACKGROUND/AIMS: Whether calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a role in regulating immunologic features of muscle cells in inflammatory environment, as it does for immune cells, remains mostly unknown. In this study, we investigated the influence of endogenous CaMKIV on the immunological characteristics of myoblasts and myotubes received IFN-γ stimulation. METHODS: C2C12 and murine myogenic precursor cells (MPCs) were cultured and differentiated in vitro, in the presence of pro-inflammatory IFN-γ. CaMKIV shRNA lentivirus transfection was performed to knockdown CaMKIV gene in C2C12 cells. pEGFP-N1-CaMKIV plasmid was delivered into knockout cells for recovering intracellular CaMKIV gene level. CREB1 antagonist KG-501 was used to block CREB signal. qPCR, immunoblot analysis, or immunofluorescence was used to detect mRNA and protein levels of CaMKIV, immuno-molecules, or pro-inflammatory cytokines and chemokines. Co-stimulatory molecules expression was assessed by FACS analysis. RESULTS: IFN-γ induces the expression or up-regulation of MHC-I/II and TLR3, and the up-regulation of CaMKIV level in muscle cells. In contrast, CaMKIV knockdown in myoblasts and myotubes leads to expression inhibition of the above immuno-molecules. As well, CaMKIV knockdown selectively inhibits pro-inflammatory cytokines/chemokines, and co-stimulatory molecules expression in IFN-γ treated myoblasts and myotubes. Finally, CaMKIV knockdown abolishes IFN-γ induced CREB pathway molecules accumulation in differentiated myotubes. CONCLUSIONS: CaMKIV can be induced to up-regulate in muscle cells under inflammatory condition, and positively mediates intrinsic immune behaviors of muscle cells triggered by IFN-γ.

Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells.

Impairment of mitochondrial biogenesis has been associated with vascular pathophysiology. The G-protein-coupled receptor (TGR5) is an important mediator of bile acid signaling and glucose metabolism. However, the effects of TGR5 on mitochondrial biogenesis in endothelial cells remain elusive. In this study, we found that activation of TGR5 using its specific agonist taurolithocholic acid (TLCA) promoted the expression of PGC-1α, a master regulator of mitochondrial biogenesis in human aortic endothelial cells (HAECs). Additionally, activation of TGR5 increased the expression of PGC-1α target genes, such as NRF1 and TFAM. Indeed, we found that TLCA treatment promoted mitochondrial biogenesis by increasing mitochondrial mass, mitochondrial-to-nuclear DNA (mtDNA/nDNA), COX-Ⅰ expression, and cytochrome c oxidase activity in HAECs. Notably, our results displayed that activation of TGR5 resulted in a functional gain in mitochondria by increasing the rate of respiration and ATP production. Mechanistically, we found that TLCA treatment activated the transcriptional factor CREB by inducing the phosphorylation of CREB at Ser133. Using the PKA/CREB inhibitor H89 abolished the effects of TLCA on PGC-1α, NRF1 and TFAM expression as well as the increase in mtDNA/nDNA and ATP production. These findings suggest that activation of TGR5 promoted mitochondrial biogenesis in endothelial cells, which is mediated by the CREB/PGC-1α signaling pathway.

Rifampicin Prevents SH-SY5Y Cells from Rotenone-Induced Apoptosis via the PI3K/Akt/GSK-3ß/CREB Signaling Pathway.

In addition to its original application for treating tuberculosis, rifampicin has multiple potential neuroprotective effects in chronic neurodegenerative diseases including Parkinson's disease (PD) and Alzheimer's disease. Inflammatory reactions and the PI3K/Akt pathway are strongly implicated in dopaminergic neuronal death in PD. This study aims to investigate whether rifampicin protects rotenone-lesioned SH-SY5Y cells via regulating PI3K/Akt/GSK-3ß/CREB pathway. Rotenone-treated SH-SY5Y cells were used as the cell model to investigate the neuroprotective effects of rifampicin. Cell viability and apoptosis of SH-SY5Y cells were determined by CCK-8 assay and flow cytometry, respectively. The expression of Akt, p-Akt, GSK-3ß, p-GSK-3ß, CREB and p-CREB were measured by Western blot. Our results showed that the cell viability and level of phospho-CREB significantly decreased in SH-SY5Y cells exposed to rotenone when compared to the control group. Both the cell viability and the expression of phospho-CREB in cells pretreated with rifampicin were higher than those of cells exposed to rotenone alone. Moreover, pretreatment of SH-SY5Y cells with rifampicin enhanced phosphorylation of Akt and suppressed activity of GSK-3ß. The addition of LY294002, a PI3K inhibitor, could suppress phosphorylation of Akt and CREB and activate GSK-3ß, resulting in abolishment of neuroprotective effects of rifampicin on cells exposed to rotenone. Rifampicin provides neuroprotection against dopaminergic degeneration, partially via the PI3K/Akt/GSK-3ß/CREB signaling pathway. These findings suggest that rifampicin could be an effective and promising neuroprotective candidate for treating PD.

Leishmania donovani Exploits Myeloid Cell Leukemia 1 (MCL-1) Protein to Prevent Mitochondria-dependent Host Cell Apoptosis.

Apoptosis is one of the mechanisms used by host cells to remove unwanted intracellular organisms, and often found to be subverted by pathogens through use of host anti-apoptotic proteins. In the present study, with the help of in vitro and in vivo approaches, we documented that the macrophage anti-apoptotic protein myeloid cell leukemia 1 (MCL-1) is exploited by the intra-macrophage parasite Leishmania donovani to protect their "home" from actinomycin D-induced mitochondria-dependent apoptosis. Among all the anti-apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both the mRNA and protein levels and compared with infected control, MCL-1-silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection-induced MCL-1 expression was regulated by transcription factor CREB (cAMP-response element-binding protein) and silencing of CREB resulted in reduced expression of MCL-1 and increased apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70-silenced macrophages, suggesting the active role of TOM70 in MCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK homo-oligomer formation thereby preventing cytochrome c release-mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited the macrophage anti-apoptotic protein MCL-1 to prevent BAK-mediated mitochondria-dependent apoptosis thereby protecting its niche, which is essential for disease progression.

Inhibitory effect of 5-iodotubercidin on pigmentation.

Melanin pigments are the primary contributors for the skin color. They are produced in melanocytes and then transferred to keratinocytes, eventually giving various colors on skin surface. Although many depigmenting and/or skin-lightening agents have been developed, there is still a growing demand on materials for reducing pigmentation. We attempted to find materials for depigmentation and/or skin-lightening using the small molecule compounds commercially available, and found that 5-iodotubercidin had inhibitory potential on pigmentation. When HM3KO melanoma cells were treated with 5-iodotubercidin, pigmentation was dramatically reduced. The 5-iodotubercidin decreased the protein level for pigmentation-related molecules such as MITF, tyrosinase, and TRP1. In addition, 5-iodotubercidin decreased the phosphorylation of CREB, while increased the phosphorylation of AKT and ERK. These data suggest that 5-iodotubercidin inhibits melanogenesis via the regulation of intracellular signaling related with pigmentation. Finally, 5-iodotubercidin markedly inhibited the melanogenesis of zebrafish embryos, an in vivo evaluation model for pigmentation. Together, these data suggest that 5-iodotubercidin can be developed as a depigmenting and/or skin-lightening agent.

TGFB1 represses the expression of SF1 and LRH1 to inhibit E2 production in rat LCs.

Leydig cells (LCs) in the adult testis have been identified as the major sites of oestrogen production, which is crucial for mammalian germ cell differentiation. Our previous work showed that transforming growth factor beta 1 (TGFB1) inhibits estradiol (E2) secretion via down-regulating Cyp19 gene expression in mature rat LCs. However, the mechanism remains unclear. In the present study, the effects of TGFB1 on the expression levels of steroidogenic factor 1 (SF1), liver receptor homolog 1 (LRH1), cAMP response element-binding protein (CREB) and cAMP responsive element modulator (CREM) were evaluated both in primary cultured LCs and in rat testis. The involvement of TGFB1 signalling in the regulation of SF1 and LRH1 expression was then validated by applying the inhibitor of the TGFB type 1 receptor (TGFBR1) SB431542. Moreover, the expression of CYP19 in testicular LCs was investigated and the production of E2 in testicular interstitial fluid (TIF) was measured. The results showed that TGFB1 especially down-regulated the expression levels of SF1 and LRH1 both in primary cultured LCs and in rat testis. The down-regulations of TGFB1 in the production of E2 in TIF and the expression of CYP19 in testicular LCs were also observed in vivo These inhibitory effects could be reversed by TGFBR1 inhibitor SB431542. Our findings suggest that TGFB1 may act through the canonical signalling pathway involving ALK5 to restrain SF1 and LRH1 accumulation and eventually attenuate Cyp19 transcription and oestrogen production in LCs.

CREB Protein Mediates Alcohol-Induced Circadian Disruption and Intestinal Permeability.

BACKGROUND: Alcoholic liver disease (ALD) is commonly associated with intestinal permeability. An unanswered question is why only a subset of heavy alcohol drinkers develop endotoxemia. Recent studies suggest that circadian disruption is the susceptibility factor for alcohol-induced gut leakiness to endotoxins. The circadian protein PER2 is increased after exposure to alcohol and siRNA knockdown of PER2 in vitro blocks alcohol-induced intestinal barrier dysfunction. We have shown that blocking CYP2E1 (i.e., important for alcohol metabolism) with siRNA inhibits the alcohol-induced increase in PER2 and suggesting that oxidative stress may mediate alcohol-induced increase in PER2 in intestinal epithelial cells. The aim of this study was to elucidate whether a mechanism incited by alcohol-derived oxidative stress mediates the transcriptional induction of PER2 and subsequent intestinal hyperpermeability. METHODS: Caco-2 cells were exposed to 0.2% alcohol with or without pretreatment with modulators of oxidative stress or PKA activity. Permeability of the Caco-2 monolayer was assessed by transepithelial electrical resistance. Protein expression was measured by Western blot and mRNA with real-time polymerase chain reaction. Wild-type C57BL/6J mice were fed with alcohol diet (29% of total calories, 4.5% v/v) for 8 weeks. Western blot was used to analyze PER2 expression in mouse proximal colon tissue. RESULTS: Alcohol increased oxidative stress, caused Caco-2 cell monolayer dysfunction, and increased levels of the circadian clock proteins PER2 and CLOCK. These effects were mitigated by pretreatment of Caco-2 cells with an antioxidant scavenger. Alcohol-derived oxidative stress activated cAMP response element-binding (CREB) via the PKA pathway and increased PER2 mRNA and protein. Inhibiting CREB prevented the increase in PER2 and Caco-2 cell monolayer hyperpermeability. CONCLUSIONS: Taken together, these data suggest that strategies to reduce alcohol-induced oxidative stress may alleviate alcohol-mediated circadian disruption and intestinal leakiness, critical drivers of ALD.