CREB-regulated Transcription Coactivator Research Articles

Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation.

Lung cancer with loss-of-function of the LKB1 tumor suppressor is a common aggressive subgroup with no effective therapies. LKB1-deficiency induces constitutive activation of cAMP/CREB-mediated transcription by a family of three CREB-regulated transcription coactivators (CRTC1-3). However, the significance and mechanism of CRTC activation in promoting the aggressive phenotype of LKB1-null cancer remain poorly characterized. Here, we observed overlapping CRTC expression patterns and mild growth phenotypes of individual CRTC-knockouts in lung cancer, suggesting functional redundancy of CRTC1-3. We consequently designed a dominant-negative mutant (dnCRTC) to block all three CRTCs to bind and co-activate CREB. Expression of dnCRTC efficiently inhibited the aberrantly activated cAMP/CREB-mediated oncogenic transcriptional program induced by LKB1-deficiency, and specifically blocked the growth of human and murine LKB1-inactivated lung cancer. Collectively, this study provides direct proof for an essential role of the CRTC-CREB activation in promoting the malignant phenotypes of LKB1-null lung cancer and proposes the CRTC-CREB interaction interface as a novel therapeutic target.

Nuts and bolts of the salt-inducible kinases (SIKs).

The salt-inducible kinases, SIK1, SIK2 and SIK3, most closely resemble the AMP-activated protein kinase (AMPK) and other AMPK-related kinases, and like these family members they require phosphorylation by LKB1 to be catalytically active. However, unlike other AMPK-related kinases they are phosphorylated by cyclic AMP-dependent protein kinase (PKA), which promotes their binding to 14-3-3 proteins and inactivation. The most well-established substrates of the SIKs are the CREB-regulated transcriptional co-activators (CRTCs), and the Class 2a histone deacetylases (HDAC4/5/7/9). Phosphorylation by SIKs promotes the translocation of CRTCs and Class 2a HDACs to the cytoplasm and their binding to 14-3-3s, preventing them from regulating their nuclear binding partners, the transcription factors CREB and MEF2. This process is reversed by PKA-dependent inactivation of the SIKs leading to dephosphorylation of CRTCs and Class 2a HDACs and their re-entry into the nucleus. Through the reversible regulation of these substrates and others that have not yet been identified, the SIKs regulate many physiological processes ranging from innate immunity, circadian rhythms and bone formation, to skin pigmentation and metabolism. This review summarises current knowledge of the SIKs and the evidence underpinning these findings, and discusses the therapeutic potential of SIK inhibitors for the treatment of disease.

The CREB Regulated Transcription Coactivator 2 Suppresses HIV-1 Transcription by Preventing RNA Pol II from Binding to HIV-1 LTR.

The CREB-regulated transcriptional co-activators (CRTCs), including CRTC1, CRTC2 and CRTC3, enhance transcription of CREB-targeted genes. In addition to regulating host gene expression in response to cAMP, CRTCs also increase the infection of several viruses. While human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter harbors a cAMP response element and activation of the cAMP pathway promotes HIV-1 transcription, it remains unknown whether CRTCs have any effect on HIV-1 transcription and HIV-1 infection. Here, we reported that CRTC2 expression was induced by HIV-1 infection, but CRTC2 suppressed HIV-1 infection and diminished viral RNA expression. Mechanistic studies revealed that CRTC2 inhibited transcription from HIV-1 LTR and diminished RNA Pol II occupancy at the LTR independent of its association with CREB. Importantly, CRTC2 inhibits the activation of latent HIV-1.Together, these data suggest that in response to HIV-1 infection, cells increase the expression of CRTC2 which inhibits HIV-1 gene expression and may play a role in driving HIV-1 into latency.

Structural Insights into the Interaction Between CRTCs and 14-3-3

The CREB-Regulated Transcriptional Coactivators (CRTCs) regulate the transcription of CREB target genes and have important functions in many biological processes. At the basal state, they are phosphorylated at multiple residues, which promotes their association with 14-3-3 that sequesters them in the cytoplasm. Upon dephosphorylation, they translocate into the nuclei and associate with CREB to activate the target gene transcription. Although three conserved serine residues in CRTCs have been implicated in their phosphorylation regulation, whether and how they mediate interactions with 14-3-3 is unclear. Here, we provide direct evidence that these residues and flanking regions interact with 14-3-3 and the structural basis of the interaction. Our study also identified a novel salt bridge in CRTC1 with an important function in binding 14-3-3, expanding the understanding of the interaction between 14-3-3 and its ligands.

The Selective SIK2 Inhibitor ARN-3236 Produces Strong Antidepressant-Like Efficacy in Mice via the Hippocampal CRTC1-CREB-BDNF Pathway.

Depression is a widespread chronic medical illness affecting thoughts, mood, and physical health. However, the limited and delayed therapeutic efficacy of monoaminergic drugs has led to intensive research efforts to develop novel antidepressants. ARN-3236 is the first potent and selective inhibitor of salt-inducible kinase 2 (SIK2). In this study, a multidisciplinary approach was used to explore the antidepressant-like actions of ARN-3236 in mice. Chronic social defeat stress (CSDS) and chronic unpredictable mild stress (CUMS) models of depression, various behavioral tests, high performance liquid chromatography-tandem mass spectrometry, stereotactic infusion, viral-mediated gene transfer, western blotting, co-immunoprecipitation and immunofluorescence were used together. It was found that ARN-3236 could penetrate the blood-brain barrier. Repeated ARN-3236 administration induced significant antidepressant-like effects in both the CSDS and CUMS models of depression, accompanied with fully preventing the stress-enhanced SIK2 expression and cytoplasmic translocation of cyclic adenosine monophosphate response element binding protein (CREB)-regulated transcription coactivator 1 (CRTC1) in the hippocampus. ARN-3236 treatment also completely reversed the down-regulating effects of CSDS and CUMS on the hippocampal brain-derived neurotrophic factor (BDNF) system and neurogenesis. Moreover, we demonstrated that the hippocampal CRTC1-CREB-BDNF pathway mediated the antidepressant-like efficacy of ARN-3236. Collectively, ARN-3236 possesses strong protecting effects against chronic stress, and could be a novel antidepressant beyond monoaminergic drugs.

Analysis of genome-wide DNA methylation patterns in obesity.

Obesity is a chronic and complex psychosomatic disease that is becoming increasingly prevalent worldwide. This study aimed to analyze whole methylation profiles to uncover the epigenetic mechanisms associated with obesity. DNA methylation profiles in blood samples from patients with obesity and normal controls were studied using the Illumina 850 K methylation microarray. The diagnostic value of the differentially methylated genes was determined using receiver operating characteristic (ROC) analysis. The expression of selected candidate genes was verified using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and pyrosequencing. A total of 9,371 significantly differentially methylated sites (7,974 hypermethylated sites and 1,397 hypomethylated sites) were identified in 4,571 genes. A difference in the distribution of differentially methylated sites (hypermethylated and hypomethylated) in both gene structures and CpG islands was observed. A total of 114 key differentially methylated sites were identified in the CpG islands. ROC results indicated that Inhibin Subunit Beta B (INHBB), Homeobox A9 (HOXA9), Troponin T3 (TNNT3), Cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB)-regulated transcription coactivator 1 (CRTC1) and Zinc finger and BTB domain-containing 7 B (ZBTB7B) could discriminate patients with obesity from normal controls. RT-qPCR results of CRTC1 and ZBTB7B were consistent with our methylation profile results. The pyrosequencing results showed that the methylation levels of CRTC1 CpG sites (CpG1 and CpG2-cg11660071) and INHBB CpG sites (CpG2) were significantly changed in patients with obesity compared with normal controls, which was consistent with our DNA methylation profile results. Our study provides new insights into the pathological mechanism of obesity.

CRTC3, a sensor and key regulator for melanogenesis, as a tunable therapeutic target for pigmentary disorders.

Background: Although CREB phosphorylation is known to be essential in UVB/cAMP-stimulated melanogenesis, CREB null mice did not show identifiable pigmentation phenotypes. Here, we show that CREB-regulated transcription co-activator 3 (CRTC3) quantitatively regulates and orchestrates melanogenesis by directly targeting microphthalmia-associated transcription factor (MITF) and regulating the expression of most key melanogenesis-related genes.Methods: We analyzed CRTC3-null, KRT14-SCF transgenic, and their crossover mice. The molecular basis of CRTC3 effects on pigmentation was investigated by histology, melanin/tyrosinase assay, immunoblotting, shRNA, promoter assay, qRT-PCR, and subcellular localization. These analyses were carried out in primary cultured melanocytes, mouse cell lines, normal human cells, co-cultures, and ex vivo human skin. CRTC/CREB activity screening was performed to identify candidate agents for the regulation of melanogenesis.Results: The coat and skin color of CRTC3-null mice was paler due to a reduction in melanin deposition. Melanogenesis-related genes were reduced in CRTC3-deficient cultured melanocytes and tail skin of CRTC3-null mice. Notably, basal levels of MITF present in CRTC3-null mice were sufficient for melanocytic differentiation/survival. Thus CRTC3-null mice showed a comparable number of epidermal melanocytes compared to control mice. Stem cell factor (SCF) introduction by crossing with KRT14-SCF mice increased epidermal melanocytes and melanin deposition in control and CRTC3-null mice, but the skin color remained still light on the CRTC3-null background. Furthermore, we identified the therapeutic potential of altiratinib to inhibit melanogenesis in human melanocytes and human skin effectively and safely.Conclusion: CRTC3 appears to be a key sensor for melanogenesis and can be used as a reversible and tunable tool for selectively regulating melanogenesis without affecting melanocyte integrity. Thus, CRTC3 can also serve as a screening tool for the discovery of ideal melanogenesis-modulating small molecules.

Perfluorooctane sulfonate (PFOS) disrupts testosterone biosynthesis via CREB/CRTC2/StAR signaling pathway in Leydig cells

Perfluorooctane sulfonate (PFOS), a stable end-product of perfluorinated compounds (PFCs), is associated with male reproductive disorders, but its underlying mechanisms are still unclear. We used in vivo and in vitro models to investigate the effects of PFOS on testosterone biosynthesis and related mechanisms. First, male ICR mice were orally administered PFOS (0−10 mg/kg/bw) for 4 weeks. Bodyweight, sperm count, reproductive hormones, mRNA expression of the genes related to testosterone biosynthesis, and the protein expression of protein kinase A (PKA), p38 mitogen-activated protein kinase (MAPK), cAMP-response element binding protein (CREB), CREB regulated transcription coactivator 2 (CRTC2) and steroidogenic acute regulatory protein (StAR) were evaluated. Furthermore, mouse primary Leydig cells were used to delineate the molecular mechanisms that mediate the effects of PFOS on testosterone biosynthesis. Our results demonstrated that PFOS dose-dependently decreased sperm count, testosterone level, CRTC2/StAR expression, and damaged testicular interstitium morphology, paralleled by increase in phosphorylated PKA, CREB and p38 in testes. Additionally, similar to the in vivo results, PFOS significantly decreased testosterone secretion, CRTC2/StAR expression, interaction between CREB and CRTC2 and binding of CREB/CRTC2 to StAR promoter region, paralleled by increase in phosphorylated-p38, PKA, and CREB expression. Meanwhile, inhibition of p38 by SB203580, or inhibition of PKA by H89 can significantly alleviate the above PFOS-induced effects. As such, the present study highlights a role of the CREB/CRTC2/StAR signaling pathway in PFOS-induced suppression of testosterone biosynthesis, advancing our understanding of molecular mechanisms for PFOS-induced male reproductive disorders

Activation and expression of endogenous CREB-regulated transcription coactivators (CRTC) 1, 2 and 3 in the rat adrenal gland.

The activation and nuclear translocation of cAMP‐response element binding protein (CREB)‐regulated transcription coactivator (CRTC)2 occurs in the rat adrenal gland, in response to adrenocorticotrophic hormone (ACTH) and stressors, and has been implicated in the transcriptional regulation of steroidogenic acute regulatory protein (StAR). We have recently demonstrated the activation of CRTC isoforms, CRTC1 and CRTC3, in adrenocortical cell lines. In the present study, we aimed to determine the activation and expression of the three CRTC isoforms in vivo in relation to Star transcription, under basal conditions and following a robust endotoxic stress challenge. Rat adrenal glands and blood plasma were collected following i.v. administration of either an ultradian‐sized pulse of ACTH or administration of lipopolysaccharide, as well as under unstressed conditions across the 24‐hour period. Plasma ACTH and corticosterone (CORT) were measured and the adrenal glands were processed for measurement of protein by western immunoblotting, RNA by a quantitative reverse transcriptase‐polymerase chain reaction and association of CRTC2 and CRTC3 with the Star promoter by chromatin immunoprecipitation. An increase in nuclear localisation of CRTC2 and CRTC3 followed increases in both ultradian and endotoxic stress‐induced plasma ACTH, and this was associated with increased CREB phosphorylation and corresponding increases in Star transcription. Both CRTC2 and CRTC3 were shown to associate with the Star promoter, with the dynamics of CRTC3 binding corresponding to that of nuclear changes in protein levels. CRTC isoforms show little variation in ultradian expression or variation across 24 hours, although evidence of long‐term down‐regulation following endotoxic stress was found. We conclude that co‐transcription factors CRTC2 and, more clearly, CRTC3 appear to act alongside phosphorylated CREB in the generation of ultradian pulses of Star transcription, essential for the maintenance of basal StAR expression. Similarly, our findings suggest CRTC2 and CRTC3 mediate Star transcriptional initiation following an endotoxic stressor; however, other transcription factors are likely to be responsible for the long‐term up‐regulation of adrenal Star transcription.

Anti-melanogenic property of ginsenoside Rf from Panax ginseng via inhibition of CREB/MITF pathway in melanocytes and ex vivo human skin

BackgroundGinsenosides of Panax ginseng are used to enhance skin health and beauty. The present study aimed to investigate the potential use of ginsenoside Rf (Rf) from Panax ginseng as a new anti-pigmentation agent. MethodsThe anti-melanogenic effects of Rf were explored. The transcriptional activity of the cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and the expression levels of tyrosinase, microphthalmia-associated transcription factor (MITF), and tyrosinase-related proteins (Tyrps) were evaluated in melanocytes and UV-irradiated ex vivo human skin. ResultsRf significantly inhibited Forskolin (FSK) or UV-stimulated melanogenesis. Consistently, cellular tyrosinase activity and levels of MITF, tyrosinase, and Tyrps were downregulated. Furthermore, Rf suppressed MITF promoter activity, which was stimulated by FSK or CREB-regulated transcription coactivator 3 (CRTC3) overexpression. Increased CREB phosphorylation and protein kinase A (PKA) activity induced by FSK were also mitigated in the presence of Rf. ConclusionRf can be used as a reliable anti-pigmentation agent, which has a scientifically confirmed and reproducible action mechanism, via inhibition of CREB/MITF pathway.

CRTC1 signaling involvement in depression-like behavior of prenatally stressed offspring rat

A large body of literature has demonstrated that prenatal stress (PS) can induce depression-like behavior in the offspring. However, the underlying mechanism remains largely unknown. CREB-regulated transcriptional coactivator 1(CRTC1) has recently been shown to involve in mood regulation. This research aims to investigate whether CRTC1 signaling was involved in the depression-like behavior of prenatally stressed offspring rats. Sucrose preference test (SPT), forced swimming test (FST) and open field test (OFT) were adopted to test the depression-like behavior in the male offspring rats, and CRTC1 signaling was measured. The results showed that there were significantly reduced sucrose intake in SPT and prolonged immobility time in FST in PS-exposure offspring rats. It was also found decreased levels of total CRTC1, nuclear CRTC1, calcineurin, brain-derived neurotrophic factor (BDNF) and c-fos, but increased cytoplasmic p-CRTC1 in the hippocampus (HIP) and prefrontal cortex (PFC) of the offspring rats. Furthermore, the mRNA level of CRTC1, calcineurin, BDNF, c-fos were down-regulated. Abnormal expression of CRTC1 signaling could be alleviated by fluoxetine treatment. In conclusion, our research indicated that the aberration of CRTC1 expression and/or phosphorylation activity might play a vital role in PS-induced depression-like behavior of offspring rats.

Imipramine exerts antidepressant-like effects in chronic stress models of depression by promoting CRTC1 expression in the mPFC

Recent studies have suggested that CREB-regulated transcription coactivator 1 (CRTC1) plays a role in the pathophysiology of depression. Although imipramine is thought to prevent the reuptake of synaptic serotonin and norepinephrine, its antidepressant-like mechanisms remain elusive. In this study, the effects of imipramine on CRTC1 were studied in several models of depression, including the chronic restraint stress (CRS), chronic unpredictable mild stress (CUMS) and chronic social defeat stress (CSDS) models. We examined whether repeated imipramine administration can reverse the effects of CRS, CUMS and CSDS on CRTC1 expression in both the hippocampus and medial prefrontal cortex (mPFC). Furthermore, genetic knockdown of CRTC1 by CRTC1-shRNA was used to determine whether CRTC1 is necessary for the antidepressant-like effects of imipramine in mice. Our results showed that imipramine reversed the down-regulating effects of CRS, CUMS and CSDS on CRTC1 expression in the mPFC but not the hippocampus, and that CRTC1-shRNA fully abolished the antidepressant-like actions of imipramine in mice. In conclusion, CRTC1 in the mPFC is involved in the antidepressant mechanism of imipramine.

Acute Nicotine Treatment Alleviates LPS-Induced Impairment of Fear Memory Reconsolidation Through AMPK Activation and CRTC1 Upregulation in Hippocampus.

BackgroundFear memory is a fundamental capability for animals and humans to survive. Its impairment results in the disability to avoid danger. When memory is reactivated, a reconsolidation process, which can be disrupted by various stimuli, including inflammation, is required to become permanent. Nicotine has been shown to improve cognitive deficits induced by inflammation and other stimuli. Therefore, in the present study, we investigated the effect of nicotine on lipopolysaccharide (LPS)-induced impairment of fear memory reconsolidation and the underlying mechanism.MethodsStep-through inhibitory avoidance task was recruited to study fear memory of rat, i.p. LPS (0.5 mg/kg) treatment was used to induce inflammation, and western blot and immunostaining were applied to detect protein expression and distribution in medial prefrontal cortex and hippocampus.ResultsOur data showed that LPS induced fear memory reconsolidation impairment without affecting retrieval. In addition, LPS significantly increased inflammation factors tumor necrosis factor-α and interleukin-1 beta and decreased CREB-regulated transcription coactivator 1 (CRTC1) expression and adenosine monophosphate-activated protein kinase (AMPK) activation in hippocampus. More importantly, LPS significantly decreased CRTC1 expression and AMPK activation in neurons by activating microglia cells. Of note, either nicotine treatment or activation of AMPK by intracerebroventricular infusion of metformin reduced LPS-induced impairment of fear memory reconsolidation and ameliorated inflammation factor tumor necrosis factor-α and interleukin-1 beta as well as the expression of CRTC1.ConclusionsIn conclusion, our results showed that acute nicotine treatment alleviates LPS-induced impairment of fear memory reconsolidation through activation of AMPK and upregulation of CRTC1 in hippocampus.

Farnesoid X Receptor-Mediated Cytoplasmic Translocation of CRTC2 Disrupts CREB-BDNF Signaling in Hippocampal CA1 and Leads to the Development of Depression-Like Behaviors in Mice.

BackgroundWe recently identified neuronal expression of farnesoid X receptor (FXR), a bile acid receptor known to impair autophagy by inhibiting cyclic adenosine monophosphate response element-binding protein (CREB), a protein whose underfunctioning is linked to neuroplasticity and depression. In this study, we hypothesize that FXR may mediate depression via a CREB-dependent mechanism.MethodsDepression was induced in male C57BL6/J mice via chronic unpredictable stress (CUS). Subjects underwent behavioral testing to identify depression-like behaviors. A variety of molecular biology techniques, including viral-mediated gene transfer, Western blot, co-immunoprecipitation, and immunofluorescence, were used to correlate depression-like behaviors with underlying molecular and physiological events.ResultsOverexpression of FXR, whose levels were upregulated by CUS in hippocampal CA1, induced or aggravated depression-like behaviors in stress-naïve and CUS-exposed mice, while FXR short hairpin RNA (shRNA) ameliorated such symptoms in CUS-exposed mice. The behavioral effects of FXR were found to be associated with changes in CREB-brain-derived neurotrophic factor (BDNF) signaling, as FXR overexpression aggravated CUS-induced reduction in BDNF levels while the use of FXR shRNA or disruption of FXR-CREB signaling reversed the CUS-induced reduction in the phosphorylated CREB and BDNF levels. Molecular analysis revealed that FXR shRNA prevented CUS-induced cytoplasmic translocation of CREB-regulated transcription coactivator 2 (CRTC2); CRTC2 overexpression and CRTC2 shRNA abrogated the regulatory effect of FXR overexpression or FXR shRNA on CUS-induced depression-like behaviors.ConclusionsIn stress conditions, increased FXR in the CA1 inhibits CREB by targeting CREB and driving the cytoplasmic translocation of CRTC2. Uncoupling of the FXR-CREB complex may be a novel strategy for depression treatment.

CRTC1 Maintains Metabolic Homeostasis and Protects Against Partial Lipodystrophy & Insulin Resistance by Regulating Diet‐Responsive White Adipose Tissue Remodeling

Humans evolved the ability to store lipid in specialized tissue called adipose to support survival. Adipose provides an energy depot that can be quickly mobilized in response to acute stress (fight‐or‐flight), for reproduction and during extended fasting. Defects in white adipose tissue (WAT) function, and inability of the WAT depots to expand in the context of overnutrition, will lead to ectopic lipid accumulation in the muscle and liver triggering serious metabolic diseases such as type 2 diabetes (T2D).Our data demonstrate that CREB Regulated Transcription Coactivator 1 (CRTC1) gene function is important in maintaining metabolic fitness. CRTC1 is expressed primarily in the hypothalamus where it controls satiety in response to cAMP and Ca2+ signals. Recent studies identified small nucleotide polymorphisms (SNPs) in the human gene locus CRTC1 that correlate with decreased adiposity in the context of obesity, indicating that CRTC1 may play an important role in adipose function. Here, we describe a previously unknown role of CRTC1 in the regulation of epididymal WAT (eWAT) expansion in response to high fat diet (HFD).Basal CRTC1 protein expression in eWAT is low compared to that in the brain, yet specifically increased in eWAT in response to HFD. Loss of CRTC1 by whole‐body germline knockout (CRTC1 KO mice), acute antisense oligonucleotide knockdown in WAT (CRTC1 ASO mice), and tissue‐specific germline knockout in adipose tissue (CRTC1 AKO mice) results in impaired HFD‐responsive eWAT expansion, observed as a ~50% decrease in eWAT size. Diminished capacity for eWAT expansion coincides with elevated lipid accumulation in liver and muscle, hyperglycemia, hyperinsulinemia and glucose intolerance in these animals relative to wildtype or control.CRTC1 knockdown by adenoviral shRNA decreased glucose uptake, increased lipolysis and impeded expression of the key adipogenesis marker PPARγ in differentiated 3T3‐L1 adipocytes. Animal models with decreased CRTC1 expression (CRTC1 KO, ASO and AKO) also exhibited decreased PPARγ expression in eWAT but only on HFD, suggesting that CRTC1 is required for diet‐responsive eWAT remodeling and for maintaining a differentiated state in adipocytes. Supportive of this hypothesis, we observed significantly decreased eWAT FGF1 mRNA expression, plasma FGF1 and WAT FGF1 levels in HFD‐fed animal models of CRTC1 loss relative to control. FGF1 plays a major role in eWAT remodeling and expansion in response to diet: our data indicate that CRTC1 is required for HFD‐mediated upregulation of FGF1. Together, our findings reveal a regulatory axis that is essential for eWAT remodeling and expansion, and that leads to ectopic lipid deposition and insulin resistance when perturbed. Understanding how CRTC1 regulates FGF1 and impacts the PPARγ pathway in eWAT will provide important insights into adipocyte function and into how eWAT expansion is modulated to maintain metabolic homeostasis, allowing identification of new potential targets to treat diseases such as T2D.Support or Funding InformationLife Science Research Foundation Fellowship, Sponsor: Gilead Sciences Inc.; R01 DK083834, PI: Marc Montminy

P7C3-A20 alleviates fatty liver by shaping gut microbiota and inducing FGF21/FGF1, via the AMP-activated protein kinase/CREB regulated transcription coactivator 2 pathway.

Non-alcoholic fatty liver disease (NAFLD) is a worldwide public health problem with no established pharmacological therapy. Here, we explored the potential benefit of P7C3-A20, a novel aminopropyl carbazole compound with neuroprotective activity, in a NAFLD model, induced in mice by a high-fat diet (HFD). C57BL/6J mice were given a HFD (42% fat content) for 16 weeks to induce NAFLD. P7C3-A20 (20 mg·kg-1 ·day-1 ) was given by gavage for 2 weeks. Indirect calorimetry, histological analysis, immunoblotting, immunohistochemistry, and biomedical examinations were performed. Gut microbiota were determined using a 16S ribosomal RNA sequencing analysis. P7C3-A20 treatment reduced body weight gain/adiposity, improved insulin resistance, promoted energy expenditure (O2 consumption/CO2 production), inhibited lipid oxidation, suppressed hepatic inflammation (Kupffer cell number and pro-inflammatory factors), decreased necroptosis/apoptosis (receptor-interacting protein kinase 3, cleaved caspase-3, and TUNEL), and alleviated liver fibrosis and injury. Mechanistically, P7C3-A20 stimulated FGF21 and FGF1 via activating liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK), which further resulted in a reduced nuclear translocation of CREB-regulated transcription coactivator 2 (CRTC2). In AMPKα2 knockout mice, the protection of P7C3-A20 against HFD-induced metabolism abnormalities and fat accumulation, as well as the elevation of blood FGF21 and FGF1, was abolished. P7C3-A20 increased the gut microbiota species richness. Moreover, it enhanced the proportions of Akkermansia, Lactobacillus, and Prevotellaceae, while reducing the proportions of Enterobacteriaceae, Escherichia, and Parasutterella. P7C3-A20 increased levels of NAD+ and alleviated NAFLD through stimulating FGF21 and FGF1 in an LKB1/AMPK/CRTC2-dependent manner and shaping gut microbiota. This article is part of a themed issue on Cellular metabolism and diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.10/issuetoc.

Zhenqing recipe attenuates non-alcoholic fatty liver disease by regulating the SIK1/CRTC2 signaling in experimental diabetic rats

BackgroundAs a compound Chinese medicine, Zhenqing Recipe (ZQR) has been shown to ameliorate hyperglycemia, hyperlipidemia, fatty liver and insulin resistance in patients with diabetes and diabetic rats. In this paper, we further examined the effect of ZQR on diabetes complicated by non-alcoholic fatty liver disease (NAFLD) and the underlying molecular mechanisms.MethodsDiabetic rats with NAFLD were developed by a high-fat diet (HFD) with low-dose streptozotocin (STZ) injection for 4 weeks. These rats were randomly separated into the diabetic model (DM), ZQR, metformin (Met), adenovirus expressing-salt-induced kinase 1 (Ad-SIK1) and adenovirus labeled with green fluorescent protein (Ad-GFP) groups. The effects on hepatic expression of gluconeogenic genes, glycolipid metabolism and pathological changes were subsequently detected.ResultsSerum glucose, triglycerides (TG), total cholesterol (TC) and hepatic TG were reduced in the ZQR group. The histopathological and immunohistochemical changes in the liver and pancreas in the ZQR group were significantly alleviated. The decrease of SIK1 expression was observed in the liver of diabetic rats induced by HFD and STZ. SIK1 overexpression in the liver relieved hyperglycemia, hyperlipidemia and fatty liver. Both the mRNA and protein levels of CREB-regulated transcription co-activator 2 (CRTC2), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in the liver were drastically reduced, whereas those of SIK1 were markedly increased in the ZQR group compared to levels in the DM group. Compared with the DM group, Ser577 phosphorylation of SIK1 was obviously reduced in the liver, while T182 phosphorylation of SIK1 and S171 phosphorylation of CRTC2 were evidently increased in the Ad-SIK1, Met and ZQR groups.ConclusionsZQR ameliorates hepatic gluconeogenesis and lipid storage in diabetic rats induced by HFD and STZ by activating the SIK1/CRTC2 signaling pathway. Upregulating hepatic SIK1 by ZQR may represent an efficient strategy for treating diabetes with NAFLD.

Genome-Wide Association Analysis Reveals Key Genes Responsible for Egg Production of Lion Head Goose.

The lion head goose is one of the most important agricultural resources in China; however, its breeding process is relatively slow. In the present study, a genome-wide association study was performed for the genetic selection of egg production characters in lion head geese. We detected 30 single-nucleotide polymorphisms located in or near 30 genes that might be associated with egg production character, and quantitative real-time polymerase chain reaction was used to verify their expression level in lion head geese. The results showed that the expression levels of CRTC1 (encoding CREB-regulated transcription coactivator 1), FAAH2 (encoding fatty acid amide hydrolase 2), GPC3 (encoding glypican 3), and SERPINC1 (encoding serpin family C member 1) in high egg production population were significantly lower than those in the low egg production populations (*P < 0.05). The expression levels of CLPB (encoding caseinolytic peptidase B protein homolog), GNA12 (encoding guanine nucleotide-binding protein subunit alpha-12), and ZMAT5 (encoding zinc finger, matrin type 5) in the high egg production population were significantly higher than those in the low egg production populations (*P < 0.05). The expression of BMP4 (encoding bone morphogenetic protein 4), FRMPD3 (encoding FERM and PDZ domain containing 3), LIF (encoding leukemia inhibitory factor), and NFYC (encoding nuclear transcription factor Y subunit gamma) in the high egg production population were very significantly lower than those in the low egg production population (**P < 0.01). Our findings provide an insight into the economic traits of lion head goose. These candidate genes might be valuable for future breeding improvement.

Phosphorylation of CREB-Specific Coactivator CRTC2 at Ser238 Promotes Proliferation, Migration, and Invasion of Colorectal Cancer Cells.

cAMP response element binding protein (CREB)-regulated transcription coactivator 2 (CRTC2), a member of the novel CRTC family of transcriptional coactivators that activates basic leucine zipper transcription factors, including CREB, is overexpressed in many carcinomas, including colon cancer. Phosphorylation of CRTC2 protein at different residues is important for its subcellular localization and activity. However, the functions of some of the serine phosphorylation sites have not been elucidated. This study aimed to investigate the effects of phosphorylation of Ser127, Ser238, and Ser245 sites of CRTC2 in colorectal cancer (CRC) cells. Recombinant lentiviral particles with a CRTC2-targeting small hairpin RNA (shRNA) sequence were transfected into CRC cells to obtained shCRTC2 cell lines. Site-directed mutagenesis of Ser127, Ser238, and Ser245 cells were constructed by transfecting CRTC2 cDNA containing S127A, S238A, and S245A mutations into shCRTC2. Cell proliferation was measured by cell counting kit-8. Cell migration and invasion were examined by transwell assay. mRNA expression was assayed by qRT-PCR, and protein expression was determined by Western blot. Our results indicate that CRTC2 is overexpressed in CRC cells. Knockdown of CRTC2 inhibits the proliferation, migration, and invasion of CRC cells. When the phosphorylation of CRTC2 Ser238 decreases due to the lack of ERK2, the phosphorylation of Ser171 site increases. The proliferation, migration and invasion of CRC cells were inhibited, the nuclear aggregation of CRTC2 in the nucleus was reduced, and the interaction between CRTC2 and CREB was weaken. It is shown that the phosphorylation of CRTC2 Ser238 is important for CREB transcriptional activity. These findings may help in the identification of potentially new targets for CRC therapy.

Role of CRTC2 in Metabolic Homeostasis: Key Regulator of Whole-Body Energy Metabolism?

Cyclic adenosine monophosphate (cAMP) signaling is critical for regulating metabolic homeostasis in mammals. In particular, transcriptional regulation by cAMP response element-binding protein (CREB) and its coactivator, CREB-regulated transcription coactivator (CRTC), is essential for controlling the expression of critical enzymes in the metabolic process, leading to more chronic changes in metabolic flux. Among the CRTC isoforms, CRTC2 is predominantly expressed in peripheral tissues and has been shown to be associated with various metabolic pathways in tissue-specific manners. While initial reports showed the physiological role of CRTC2 in regulating gluconeogenesis in the liver, recent studies have further delineated the role of this transcriptional coactivator in the regulation of glucose and lipid metabolism in various tissues, including the liver, pancreatic islets, endocrine tissues of the small intestines, and adipose tissues. In this review, we discuss recent studies that have utilized knockout mouse models to delineate the role of CRTC2 in the regulation of metabolic homeostasis.