The nutrient sensor CRTC and Sarcalumenin/thinman represent an alternate pathway in cardiac hypertrophy.

CREB-regulated transcription co-activator (CRTC) is activated by Calcineurin (CaN) to regulate gluconeogenic genes. CaN also has roles in cardiac hypertrophy. Here, we explore a cardiac-autonomous role for CRTC in cardiac hypertrophy. In Drosophila, CRTC mutants exhibit severe cardiac restriction, myofibrillar disorganization, fibrosis, and tachycardia. Cardiac-specific CRTC knockdown (KD) phenocopies mutants, and cardiac overexpression causes hypertrophy. CaN-induced hypertrophy in Drosophila is reduced in CRTC mutants, suggesting that CRTC mediates the effects. RNA sequencing (RNA-seq) of CRTC-KD and -overexpressing hearts reveals contraregulation of metabolic genes. Genes with conserved CREB sites include the fly ortholog of Sarcalumenin, a Ca2+-binding protein. Cardiac manipulation of this gene recapitulates the CRTC-KD and -overexpression phenotypes. CRTC KD in zebrafish also causes cardiac restriction, and CRTC KD in human induced cardiomyocytes causes a reduction in Srl expression and increased action potential duration. Our data from three model systems suggest that CaN-CRTC-Sarcalumenin signaling represents an alternate, conserved pathway underlying cardiac function and hypertrophy.

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.

Inositol-1,4,5-trisphosphate receptor regulates hepatic gluconeogenesis in fasting and diabetes.

In the fasted state, increases in circulating glucagon promote hepatic glucose production through induction of the gluconeogenic program. Triggering of the cyclic AMP pathway increases gluconeogenic gene expression via the de-phosphorylation of the CREB co-activator CRTC2 (ref. 1). Glucagon promotes CRTC2 dephosphorylation in part through the protein kinase A (PKA)-mediated inhibition of the CRTC2 kinase SIK2. A number of Ser/Thr phosphatases seem to be capable of dephosphorylating CRTC2 (refs 2, 3), but the mechanisms by which hormonal cues regulate these enzymes remain unclear. Here we show in mice that glucagon stimulates CRTC2 dephosphorylation in hepatocytes by mobilizing intracellular calcium stores and activating the calcium/calmodulin-dependent Ser/Thr-phosphatase calcineurin (also known as PP3CA). Glucagon increased cytosolic calcium concentration through the PKA-mediated phosphorylation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs), which associate with CRTC2. After their activation, InsP(3)Rs enhanced gluconeogenic gene expression by promoting the calcineurin-mediated dephosphorylation of CRTC2. During feeding, increases in insulin signalling reduced CRTC2 activity via the AKT-mediated inactivation of InsP(3)Rs. InsP(3)R activity was increased in diabetes, leading to upregulation of the gluconeogenic program. As hepatic downregulation of InsP(3)Rs and calcineurin improved circulating glucose levels in insulin resistance, these results demonstrate how interactions between cAMP and calcium pathways at the level of the InsP(3)R modulate hepatic glucose production under fasting conditions and in diabetes.

CRTC3 links catecholamine signalling to energy balance.

The adipose-derived hormone leptin maintains energy balance in part through central nervous system-mediated increases in sympathetic outflow that enhance fat burning. Triggering of ß-adrenergic receptors in adipocytes stimulates energy expenditure by cyclic AMP (cAMP)-dependent increases in lipolysis and fatty-acid oxidation. Although the mechanism is unclear, catecholamine signalling is thought to be disrupted in obesity, leading to the development of insulin resistance. Here we show that the cAMP response element binding (CREB) coactivator Crtc3 promotes obesity by attenuating ß-adrenergic receptor signalling in adipose tissue. Crtc3 was activated in response to catecholamine signals, when it reduced adenyl cyclase activity by upregulating the expression of Rgs2, a GTPase-activating protein that also inhibits adenyl cyclase activity. As a common human CRTC3 variant with increased transcriptional activity is associated with adiposity in two distinct Mexican-American cohorts, these results suggest that adipocyte CRTC3 may play a role in the development of obesity in humans.

The insulin-regulated CREB coactivator TORC promotes stress resistance in Drosophila.

In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein (CREB) coactivator transducer of regulated CREB activity 2 (TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1. Here we show that starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augmented TORC activity in adult flies, our results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance.

NF-kappaB is a negative regulator of IL-1beta secretion as revealed by genetic and pharmacological inhibition of IKKbeta.

IKKbeta-dependent NF-kappaB activation plays a key role in innate immunity and inflammation, and inhibition of IKKbeta has been considered as a likely anti-inflammatory therapy. Surprisingly, however, mice with a targeted IKKbeta deletion in myeloid cells are more susceptible to endotoxin-induced shock than control mice. Increased endotoxin susceptibility is associated with elevated plasma IL-1beta as a result of increased pro-IL-1beta processing, which was also seen upon bacterial infection. In macrophages enhanced pro-IL-1beta processing depends on caspase-1, whose activation is inhibited by NF-kappaB-dependent gene products. In neutrophils, however, IL-1beta secretion is caspase-1 independent and depends on serine proteases, whose activity is also inhibited by NF-kappaB gene products. Prolonged pharmacologic inhibition of IKKbeta also augments IL-1beta secretion upon endotoxin challenge. These results unravel an unanticipated role for IKKbeta-dependent NF-kappaB signaling in the negative control of IL-1beta production and highlight potential complications of long-term IKKbeta inhibition.

The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9.

Here we have identified 'triple D' (3d), a recessive N-ethyl-N-nitrosourea-induced mutation and phenotype in which no signaling occurs via the intracellular Toll-like receptors 3, 7 and 9 (sensors for double-stranded RNA, single-stranded RNA and unmethylated DNA, respectively). The 3d mutation also prevented cross-presentation and diminished major histocompatibility complex class II presentation of exogenous antigen; it also caused hypersusceptibility to infection by mouse cytomegalovirus and other microbes. By positional identification, we found 3d to be a missense allele of Unc93b1, which encodes the 12-membrane-spanning protein UNC-93B, a highly conserved molecule found in the endoplasmic reticulum with multiple paralogs in mammals. Innate responses to nucleic acids and exogenous antigen presentation, which both initiate in endosomes, thus seem to depend on an endoplasmic reticulum-resident protein, which suggests communication between these organellar systems.

Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection.

Several subsets of dendritic cells have been shown to produce type I IFN in response to viral infections, thereby assisting the natural killer cell-dependent response that eliminates the pathogen. Type I IFN production can be induced both by unmethylated CpG-oligodeoxynucleotide and by double-stranded RNA. Here, we describe a codominant CpG-ODN unresponsive phenotype that results from an N-ethyl-N-nitrosourea-induced missense mutation in the Tlr9 gene (Tlr9(CpG1)). Mice homozygous for the Tlr9(CpG1) allele are highly susceptible to mouse cytomegalovirus infection and show impaired infection-induced secretion of IFN-alpha/beta and natural killer cell activation. We also demonstrate that both the Toll-like receptor (TLR) 9 --> MyD88 and TLR3 --> Trif signaling pathways are activated in vivo on viral inoculation, and that each pathway contributes to innate defense against systemic viral infection. Whereas both pathways lead to type I IFN production, neither pathway offers full protection against mouse cytomegalovirus infection in the absence of the other. The Tlr9(CpG1) mutation alters a leucine-rich repeat motif and lies within a receptor domain that is conserved within the evolutionary cluster encompassing TLRs 7, 8, and 9. In other TLRs, including three mouse-specific TLRs described in this paper, the affected region is not represented. The phenotypic effect of the Tlr9(CpG1) allele thus points to a critical role for TLR9 in viral sensing and identifies a vulnerable amino acid within the ectodomain of three TLR proteins, essential for a ligand response.

Lps2: a new locus required for responses to lipopolysaccharide, revealed by germline mutagenesis and phenotypic screening.

Both forward and reverse genetic techniques have been used to define components of the mammalian lipopolysaccharide (LPS) receptor. TLR4, identified by a forward genetic approach as the product of the classical Lps locus, is the only known transmembrane component of the mammalian LPS receptor. Gene knockout work has also established that LPS signal transduction requires the integrity of CD14, MD-2, and, in part, MyD88, IRAK4, and TRAF-6. However, there is no reason to believe that these are the only proteins that make up the receptor/transducer apparatus. To examine the possibility that other proteins may be involved, we initiated a mutagenesis program, in which germline mutations are induced in mice using N-ethyl-N-nitrosourea (ENU), and macrophages from individual animals are screened for their competence to respond to LPS. We now report the existence of a new locus, Lps2, which is required for TNF production in response to LPS. The Lps2 mutation that we have identified is co-dominant, is similar in phenotypic effect to Lpsd, and does not represent a novel allele of any of the genes that are known to encode the 'core' LPS signaling apparatus. The Lps2 mutation does not preclude signaling initiated by peptidoglycan or unmethylated DNA. Hence, genetic data suggest that there is at least one 'missing' component of the LPS receptor complex that has yet to be found.