Hepatic noradrenergic innervation acts via CREB/CRTC2 to activate gluconeogenesis during cold.

BACKGROUND AND AIM: Although it is well established that hormones like glucagon stimulates gluconeogenesis via the PKA-mediated phosphorylation of CREB and dephosphorylation of the cAMP-regulated CREB coactivators CRTC2, the role of neural signals in the regulation of gluconeogenesis remains uncertain. METHODS AND RESULTS: Here, we characterize the noradrenergic bundle architecture in mouse liver; we show that the sympathoexcitation induced by acute cold exposure promotes hyperglycemia and upregulation of gluconeogenesis via triggering of the CREB/CRTC2 pathway. Following its induction by dephosphorylation, CRTC2 translocates to the nucleus and drives the transcription of key gluconeogenic genes. Rodents submitted to different models of sympathectomy or knockout of CRTC2 do not activate gluconeogenesis in response to cold. Norepinephrine directly acts in hepatocytes mainly through a Ca2+-dependent pathway that stimulates CREB/CRTC2, leading to activation of the gluconeogenic program. CONCLUSION: Our data demonstrate the importance of the CREB/CRTC2 pathway in mediating effects of hepatic sympathetic inputs on glucose homeostasis, providing new insights into the role of norepinephrine in health and disease.

The nutrient sensor CRTC & Sarcalumenin / Thinman represent a new pathway in cardiac hypertrophy.

Obesity and type 2 diabetes are at epidemic levels and a significant proportion of these patients are diagnosed with left ventricular hypertrophy. CREB R egulated T ranscription C o-activator ( CRTC ) is a key regulator of metabolism in mammalian hepatocytes, where it is activated by calcineurin (CaN) to increase expression of gluconeogenic genes. CaN is known its role in pathological cardiac hypertrophy, however, a role for CRTC in the heart has not been identified. In Drosophila , CRTC null mutants have little body fat and exhibit severe cardiac restriction, myofibrillar disorganization, cardiac fibrosis and tachycardia, all hallmarks of heart disease. Cardiac-specific knockdown of CRTC , or its coactivator CREBb , mimicked the reduced body fat and heart defects of CRTC null mutants. Comparative gene expression in CRTC loss- or gain-of-function fly hearts revealed contra-regulation of genes involved in glucose, fatty acid, and amino acid metabolism, suggesting that CRTC also acts as a metabolic switch in the heart. Among the contra-regulated genes with conserved CREB binding sites, we identified the fly ortholog of Sarcalumenin, which is a Ca 2+ -binding protein in the sarcoplasmic reticulum. Cardiac knockdown recapitulated the loss of CRTC cardiac restriction and fibrotic phenotypes, suggesting it is a downstream effector of CRTC we named thinman ( tmn ). Importantly, cardiac overexpression of either CaN or CRTC in flies caused hypertrophy that was reversed in a CRTC mutant background, suggesting CRTC mediates hypertrophy downstream of CaN, perhaps as an alternative to NFAT. CRTC novel role in the heart is likely conserved in vertebrates as knockdown in zebrafish also caused cardiac restriction, as in fl ies. These data suggest that CRTC is involved in myocardial cell maintenance and that CaN-CRTC- Sarcalumenin/ tmn signaling represents a novel and conserved pathway underlying cardiac hypertrophy.

Activation of the adipocyte CREB/CRTC pathway in obesity.

Obesity is a major risk factor for the development of type II diabetes. Increases in adipose tissue mass trigger insulin resistance via the release of pro-inflammatory cytokines from adipocytes and macrophages. CREB and the CRTC coactivators have been found to promote insulin resistance in obesity, although the mechanism is unclear. Here we show that high fat diet feeding activates the CREB/CRTC pathway in adipocytes by decreasing the expression of SIK2, a Ser/Thr kinase that phosphorylates and inhibits CRTCs. SIK2 levels are regulated by the adipogenic factor C/EBPα, whose expression is reduced in obesity. Exposure to PPARγ agonist rescues C/EBPα expression and restores SIK2 levels. CRTC2/3 promote insulin resistance via induction of the chemokines CXCL1/2. Knockout of CRTC2/3 in adipocytes reduces CXCL1/2 expression and improves insulin sensitivity. As administration of CXCL1/2 reverses salutary effects of CRTC2/3 depletion, our results demonstrate the importance of the CREB/CRTC pathway in modulating adipose tissue function.

Transcriptional co-activator regulates melanocyte differentiation and oncogenesis by integrating cAMP and MAPK/ERK pathways.

The cyclic AMP pathway promotes melanocyte differentiation by activating CREB and the cAMP-regulated transcription co-activators 1-3 (CRTC1-3). Differentiation is dysregulated in melanomas, although the contributions of CRTC proteins is unclear. We report a selective differentiation impairment in CRTC3 KO melanocytes and melanoma cells, due to downregulation of oculo-cutaneous albinism II (OCA2) and block of melanosome maturation. CRTC3 stimulates OCA2 expression by binding to CREB on a conserved enhancer, a regulatory site for pigmentation and melanoma risk. CRTC3 is uniquely activated by ERK1/2-mediated phosphorylation at Ser391 and by low levels of cAMP. Phosphorylation at Ser391 is constitutively elevated in human melanoma cells with hyperactivated ERK1/2 signaling; knockout of CRTC3 in this setting impairs anchorage-independent growth, migration, and invasiveness, whereas CRTC3 overexpression supports cell survival in response to the mitogen-activated protein kinase (MAPK) inhibitor vemurafenib. As melanomas expressing gain-of-function mutations in CRTC3 are associated with reduced survival, our results suggest that CRTC3 inhibition may provide therapeutic benefit in this setting.

Crtc modulates fasting programs associated with 1-C metabolism and inhibition of insulin signaling.

Fasting in mammals promotes increases in circulating glucagon and decreases in circulating insulin that stimulate catabolic programs and facilitate a transition from glucose to lipid burning. The second messenger cAMP mediates effects of glucagon on fasting metabolism, in part by promoting the phosphorylation of CREB and the dephosphorylation of the cAMP-regulated transcriptional coactivators (CRTCs) in hepatocytes. In Drosophila, fasting also triggers activation of the single Crtc homolog in neurons, via the PKA-mediated phosphorylation and inhibition of salt-inducible kinases. Crtc mutant flies are more sensitive to starvation and oxidative stress, although the underlying mechanism remains unclear. Here we use RNA sequencing to identify Crtc target genes that are up-regulated in response to starvation. We found that Crtc stimulates a subset of fasting-inducible genes that have conserved CREB binding sites. In keeping with its role in the starvation response, Crtc was found to induce the expression of genes that inhibit insulin secretion (Lst) and insulin signaling (Impl2). In parallel, Crtc also promoted the expression of genes involved in one-carbon (1-C) metabolism. Within the 1-C pathway, Crtc stimulated the expression of enzymes that encode modulators of S-adenosyl-methionine metabolism (Gnmt and Sardh) and purine synthesis (ade2 and AdSl) Collectively, our results point to an important role for the CREB/CRTC pathway in promoting energy balance in the context of nutrient stress.

The KLDpT activation loop motif is critical for MARK kinase activity.

MAP/microtubule-affinity regulating kinases (MARK1-4) are members of the AMPK family of Ser/Thr-specific kinases, which phosphorylate substrates at consensus LXRXXSXXXL motifs. Within microtubule-associated proteins, MARKs also mediate phosphorylation of variant KXGS or ζXKXGSXXNΨ motifs, interfering with the ability of tau and MAP2/4 to bind to microtubules. Here we show that, although MARKs and the closely related salt-inducible kinases (SIKs) phosphorylate substrates with consensus AMPK motifs comparably, MARKs are more potent in recognizing variant ζXKXGSXXNΨ motifs on cellular tau. In studies to identify regions of MARKs that confer catalytic activity towards variant sites, we found that the C-terminal kinase associated-1 (KA1) domain in MARK1-3 mediates binding to microtubule-associated proteins CLASP1/2; but this interaction is dispensable for ζXKXGSXXNΨ phosphorylation. Mutational analysis of MARK2 revealed that the N-terminal kinase domain of MARK2 is sufficient for phosphorylation of both consensus and variant ζXKXGSXXNΨ sites. Within this domain, the KLDpT activation loop motif promotes MARK2 activity both intracellularly and in vitro, but has no effect on SIK2 activity. As KLDpT is conserved in all vertebrates MARKs, we conclude that this sequence is crucial for MARK-dependent regulation of cellular polarity.

The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non-small cell lung cancer.

The LKB1 tumor suppressor is often mutationally inactivated in non-small cell lung cancer (NSCLC). LKB1 phosphorylates and activates members of the AMPK family of Ser/Thr kinases. Within this family, the salt-inducible kinases (SIKs) modulate gene expression in part via the inhibitory phosphorylation of the CRTCs, coactivators for CREB (cAMP response element-binding protein). The loss of LKB1 causes SIK inactivation and the induction of the CRTCs, leading to the up-regulation of CREB target genes. We identified CRTC2 as a critical factor in LKB1-deficient NSCLC. CRTC2 is unphosphorylated and therefore constitutively activated in LKB1-mutant NSCLC, where it promotes tumor growth, in part via the induction of the inhibitor of DNA binding 1 (ID1), a bona fide CREB target gene. As ID1 expression is up-regulated and confers poor prognosis in LKB1-deficient NSCLC, our results suggest that small molecules that inhibit CRTC2 and ID1 activity may provide therapeutic benefit to individuals with NSCLC.

CREB Promotes Beta Cell Gene Expression by Targeting Its Coactivators to Tissue-Specific Enhancers.

CREB mediates effects of cyclic AMP on cellular gene expression. Ubiquitous CREB target genes are induced following recruitment of CREB and its coactivators to promoter proximal binding sites. We found that CREB stimulates the expression of pancreatic beta cell-specific genes by targeting CBP/p300 to promoter-distal enhancer regions. Subsequent increases in histone acetylation facilitate recruitment of the coactivators CRTC2 and BRD4, leading to release of RNA polymerase II over the target gene body. Indeed, CREB-induced hyperacetylation of chromatin over superenhancers promoted beta cell-restricted gene expression, which is sensitive to inhibitors of CBP/p300 and BRD4 activity. Neurod1 appears critical in establishing nucleosome-free regions for recruitment of CREB to beta cell-specific enhancers. Deletion of a CREB-Neurod1-bound enhancer within the Lrrc10b-Syt7 superenhancer disrupted the expression of both genes and decreased beta cell function. Our results demonstrate how cross talk between signal-dependent and lineage-determining factors promotes the expression of cell-type-specific gene programs in response to extracellular cues.

Adaptive Transcriptional Responses by CRTC Coactivators in Cancer.

Adaptive stress signaling networks directly influence tumor development and progression. These pathways mediate responses that allow cancer cells to cope with both tumor cell-intrinsic and cell-extrinsic insults and develop acquired resistance to therapeutic interventions. This is mediated in part by constant oncogenic rewiring at the transcriptional level by integration of extracellular cues that promote cell survival and malignant transformation. The cAMP-regulated transcriptional coactivators (CRTCs) are a newly discovered family of intracellular signaling integrators that serve as the conduit to the basic transcriptional machinery to regulate a host of adaptive response genes. Thus, somatic alterations that lead to CRTC activation are emerging as key driver events in the development and progression of many tumor subtypes.

Mitogenic Signals Stimulate the CREB Coactivator CRTC3 through PP2A Recruitment.

The second messenger 3',5'-cyclic adenosine monophosphate (cAMP) stimulates gene expression via the cAMP-regulated transcriptional coactivator (CRTC) family of cAMP response element-binding protein coactivators. In the basal state, CRTCs are phosphorylated by salt-inducible kinases (SIKs) and sequestered in the cytoplasm by 14-3-3 proteins. cAMP signaling inhibits the SIKs, leading to CRTC dephosphorylation and nuclear translocation. Here we show that although all CRTCs are regulated by SIKs, their interactions with Ser/Thr-specific protein phosphatases are distinct. CRTC1 and CRTC2 associate selectively with the calcium-dependent phosphatase calcineurin, whereas CRTC3 interacts with B55 PP2A holoenzymes via a conserved PP2A-binding region (amino acids 380-401). CRTC3-PP2A complex formation was induced by phosphorylation of CRTC3 at S391, facilitating the subsequent activation of CRTC3 by dephosphorylation at 14-3-3 binding sites. As stimulation of mitogenic pathways promoted S391 phosphorylation via the activation of ERKs and CDKs, our results demonstrate how a ubiquitous phosphatase enables cross talk between growth factor and cAMP signaling pathways at the level of a transcriptional coactivator.

cAMP-inducible coactivator CRTC3 attenuates brown adipose tissue thermogenesis.

In response to cold exposure, placental mammals maintain body temperature by increasing sympathetic nerve activity in brown adipose tissue (BAT). Triggering of ß-adrenergic receptors on brown adipocytes stimulates thermogenesis via induction of the cAMP/PKA pathway. Although cAMP response element-binding protein (CREB) and its coactivators-the cAMP-regulated transcriptional coactivators (CRTCs)-mediate transcriptional effects of cAMP in most tissues, other transcription factors such as ATF2 appear critical for induction of thermogenic genes by cAMP in BAT. Brown adipocytes arise from Myf5-positive mesenchymal cells under the control of PRDM16, a coactivator that concurrently represses differentiation along the skeletal muscle lineage. Here, we show that the CREB coactivator CRTC3 is part of an inhibitory feedback pathway that antagonizes PRDM16-dependent differentiation. Mice with a knockout of CRTC3 in BAT (BKO) have increased cold tolerance and reduced adiposity, whereas mice overexpressing constitutively active CRTC3 in adipose tissue are more cold sensitive and have greater fat mass. CRTC3 reduced sympathetic nerve activity in BAT by up-regulating the expression of miR-206, a microRNA that promotes differentiation along the myogenic lineage and that we show here decreases the expression of VEGFA and neurotrophins critical for BAT innervation and vascularization. Sympathetic nerve activity to BAT was enhanced in BKO mice, leading to increases in catecholamine signaling that stimulated energy expenditure. As reexpression of miR-206 in BAT from BKO mice reversed the salutary effects of CRTC3 depletion on cold tolerance, our studies suggest that small-molecule inhibitors against this coactivator may provide therapeutic benefit to overweight individuals.

14-3-3 proteins mediate inhibitory effects of cAMP on salt-inducible kinases (SIKs).

The salt-inducible kinase (SIK) family regulates cellular gene expression via the phosphorylation of cAMP-regulated transcriptional coactivators (CRTCs) and class IIA histone deacetylases, which are sequestered in the cytoplasm by phosphorylation-dependent 14-3-3 interactions. SIK activity toward these substrates is inhibited by increases in cAMP signaling, although the underlying mechanism is unclear. Here, we show that the protein kinase A (PKA)-dependent phosphorylation of SIKs inhibits their catalytic activity by inducing 14-3-3 protein binding. SIK1 and SIK3 contain two functional PKA/14-3-3 sites, while SIK2 has four. In keeping with the dimeric nature of 14-3-3s, the presence of multiple binding sites within target proteins dramatically increases binding affinity. As a result, loss of a single 14-3-3-binding site in SIK1 and SIK3 abolished 14-3-3 association and rendered them insensitive to cAMP. In contrast, mutation of three sites in SIK2 was necessary to fully block cAMP regulation. Superimposed on the effects of PKA phosphorylation and 14-3-3 association, an evolutionary conserved domain in SIK1 and SIK2 (the so called RK-rich region; 595-624 in hSIK2) is also required for the inhibition of SIK2 activity. Collectively, these results point to a dual role for 14-3-3 proteins in repressing a family of Ser/Thr kinases as well as their substrates.

CREB coactivators CRTC2 and CRTC3 modulate bone marrow hematopoiesis.

Populations of circulating immune cells are maintained in equilibrium through signals that enhance the retention or egress of hematopoietic stem cells (HSCs) from bone marrow (BM). Prostaglandin E2 (PGE2) stimulates HSC renewal and engraftment through, for example, induction of the cAMP pathway. Triggering of PGE2 receptors increases HSC survival in part via the PKA-mediated induction of the cAMP response element-binding protein (CREB) signaling pathway. PKA stimulates cellular gene expression by phosphorylating CREB at Ser133 and by promoting the dephosphorylation of the cAMP- responsive transcriptional coactivators (CRTCs). We show here that disruption of both CRTC2 and CRTC3 causes embryonic lethality, and that a single allele of either CRTC2 or CRTC3 is sufficient for viability. CRTC2 knockout mice that express one CRTC3 allele (CRTC2/3m mice) develop neutrophilia and splenomegaly in adulthood due to the up-regulation of granulocyte-colony stimulating factor (G-CSF); these effects are reversed following administration of neutralizing anti-G-CSF antiserum. Adoptive transfer of CRTC2/3m BM conferred the splenomegaly/neutrophilia phenotype in WT recipients. Targeted disruption of both CRTC2 and CRTC3 in stromal cells with a mesenchymal Prx1-Cre transgene also promoted this phenotype. Depletion of CRTC2/3 was found to decrease the expression of Suppressor of Cytokine Signaling 3 (SOCS3), leading to increases in STAT3 phosphorylation and to the induction of CEBPß, a key regulator of the G-CSF gene. As small molecule inhibition of JAK activity disrupted CEBPß induction and reduced G-CSF expression in CRTC2/3m stromal cells, our results demonstrate how cross-coupling between the CREB/CRTC and JAK/STAT pathways contributes to BM homeostasis.

Analysis of a cAMP regulated coactivator family reveals an alternative phosphorylation motif for AMPK family members.

The second messenger cAMP stimulates cellular gene expression via the PKA-mediated phosphorylation of the transcription factor CREB and through dephosphorylation of the cAMP-responsive transcriptional coactivators (CRTCs). Under basal conditions, CRTCs are phosphorylated by members of the AMPK family of Ser/Thr kinases and sequestered in the cytoplasm via a phosphorylation-dependent association with 14-3-3 proteins. Increases in cAMP promote the dephosphorylation and nuclear translocation of CRTCs, where they bind to CREB and stimulate relevant target genes. Although they share considerable sequence homology, members of the CRTC family exert non-overlapping effects on cellular gene expression through as yet unidentified mechanisms. Here we show that the three CRTCs exhibit distinct patterns of 14-3-3 binding at three conserved sites corresponding to S70, S171, and S275 (in CRTC2). S171 functions as the gatekeeper site for 14-3-3 binding; it acts cooperatively with S275 in stabilizing this interaction following its phosphorylation by the cAMP-responsive SIK and the cAMP-nonresponsive MARK kinases. Although S171 contains a consensus recognition site for phosphorylation by AMPK family members, S70 and S275 carry variant motifs (MNTGGS275LPDL), lacking basic residues that are otherwise critical for SIK/MARK recognition as well as 14-3-3 binding. Correspondingly, the activity of these motifs differs between CRTC family members. As the variant (SLPDL) motif is present and apparently phosphorylated in other mammalian proteins, our studies suggest that the regulation of cellular targets by AMPK family members is more extensive than previously appreciated.

Neuronal energy-sensing pathway promotes energy balance by modulating disease tolerance.

The starvation-inducible coactivator cAMP response element binding protein (CREB)-cAMP-regulated transcription coactivator (Crtc) has been shown to promote starvation resistance in Drosophila by up-regulating CREB target gene expression in neurons, although the underlying mechanism is unclear. We found that Crtc and its binding partner CREB enhance energy homeostasis by stimulating the expression of short neuropeptide F (sNPF), an ortholog of mammalian neuropeptide Y, which we show here is a direct target of CREB and Crtc. Neuronal sNPF was found to promote energy homeostasis via gut enterocyte sNPF receptors, which appear to maintain gut epithelial integrity. Loss of Crtc-sNPF signaling disrupted epithelial tight junctions, allowing resident gut flora to promote chronic increases in antimicrobial peptide (AMP) gene expression that compromised energy balance. Growth on germ-free food reduced AMP gene expression and rescued starvation sensitivity in Crtc mutant flies. Overexpression of Crtc or sNPF in neurons of wild-type flies dampens the gut immune response and enhances starvation resistance. Our results reveal a previously unidentified tolerance defense strategy involving a brain-gut pathway that maintains homeostasis through its effects on epithelial integrity.

Role of the cAMP Pathway in Glucose and Lipid Metabolism.

3'-5'-Cyclic adenosine monophosphate (cyclic AMP or cAMP) was first described in 1957 as an intracellular second messenger mediating the effects of glucagon and epinephrine on hepatic glycogenolysis (Berthet et al., J Biol Chem 224(1):463-475, 1957). Since this initial characterization, cAMP has been firmly established as a versatile molecular signal involved in both central and peripheral regulation of energy homeostasis and nutrient partitioning. Many of these effects appear to be mediated at the transcriptional level, in part through the activation of the transcription factor CREB and its coactivators. Here we review current understanding of the mechanisms by which the cAMP signaling pathway triggers metabolic programs in insulin-responsive tissues.

CREB pathway links PGE2 signaling with macrophage polarization.

Obesity is thought to promote insulin resistance in part via activation of the innate immune system. Increases in proinflammatory cytokine production by M1 macrophages inhibit insulin signaling in white adipose tissue. In contrast, M2 macrophages have been found to enhance insulin sensitivity in part by reducing adipose tissue inflammation. The paracrine hormone prostaglandin E2 (PGE2) enhances M2 polarization in part through activation of the cAMP pathway, although the underlying mechanism is unclear. Here we show that PGE2 stimulates M2 polarization via the cyclic AMP-responsive element binding (CREB)-mediated induction of Krupple-like factor 4 (KLF4). Targeted disruption of CREB or the cAMP-regulated transcriptional coactivators 2 and 3 (CRTC2/3) in macrophages down-regulated M2 marker gene expression and promoted insulin resistance in the context of high-fat diet feeding. As re-expression of KLF4 rescued M2 marker gene expression in CREB-depleted cells, our results demonstrate the importance of the CREB/CRTC pathway in maintaining insulin sensitivity in white adipose tissue via its effects on the innate immune system.

Hepatic Insulin Resistance Following Chronic Activation of the CREB Coactivator CRTC2.

Under fasting conditions, increases in circulating concentrations of glucagon maintain glucose homeostasis via the induction of hepatic gluconeogenesis. Triggering of the cAMP pathway in hepatocytes stimulates the gluconeogenic program via the PKA-mediated phosphorylation of CREB and dephosphorylation of the cAMP-regulated CREB coactivators CRTC2 and CRTC3. In parallel, decreases in circulating insulin also increase gluconeogenic gene expression via the de-phosphorylation and activation of the forkhead transcription factor FOXO1. Hepatic gluconeogenesis is increased in insulin resistance where it contributes to the attendant hyperglycemia. Whether selective activation of the hepatic CREB/CRTC pathway is sufficient to trigger metabolic changes in other tissues is unclear, however. Modest hepatic expression of a phosphorylation-defective and therefore constitutively active CRTC2S171,275A protein increased gluconeogenic gene expression under fasting as well as feeding conditions. Circulating glucose concentrations were constitutively elevated in CRTC2S171,275A-expressing mice, leading to compensatory increases in circulating insulin concentrations that enhance FOXO1 phosphorylation. Despite accompanying decreases in FOXO1 activity, hepatic gluconeogenic gene expression remained elevated in CRTC2S171,275A mice, demonstrating that chronic increases in CRTC2 activity in the liver are indeed sufficient to promote hepatic insulin resistance and to disrupt glucose homeostasis.

The CREB/CRTC2 pathway modulates autoimmune disease by promoting Th17 differentiation.

Following their activation in response to inflammatory signals, innate immune cells secrete T-cell-polarizing cytokines that promote the differentiation of naive CD4 T cells into T helper (Th) cell subsets. Among these, Th17 cells play a prominent role in the development of a number of autoimmune diseases. Although regarded primarily as an immunosuppressant signal, cAMP has been found to mediate pro-inflammatory effects of macrophage-derived prostaglandin E2 (PGE2) on Th17 cells. Here we show that PGE2 enhances Th17 cell differentiation via the activation of the CREB co-activator CRTC2. Following its dephosphorylation, CRTC2 stimulates the expression of the cytokines IL-17A and IL-17F by binding to CREB over both promoters. CRTC2-mutant mice have decreased Th17 cell numbers, and they are protected from experimental autoimmune encephalitis, a model for multiple sclerosis. Our results suggest that small molecule inhibitors of CRTC2 may provide therapeutic benefit to individuals with autoimmune disease.