The activity of the Epithelial Na
+
Channel (ENaC) in renal principal cells (PC) fine-tunes sodium excretion and consequently, affects blood pressure. The Gs-adenylyl cyclase-cAMP signal transduction ...pathway is believed to play a central role in the normal control of ENaC activity in PCs. The current study quantifies the importance of this signaling pathway to the regulation of ENaC activity
in vivo
using a knock-in mouse that has conditional expression of Gs-DREADD (designer receptors exclusively activated by designer drugs; GsD) in renal PCs. The GsD mouse also contains a cAMP response element-luciferase reporter transgene for non-invasive bioluminescence monitoring of cAMP signaling. Clozapine N-oxide (CNO) was used to selectively and temporally stimulate GsD. Treatment with CNO significantly increased luciferase bioluminescence in the kidneys of PC-specific GsD but not control mice. CNO also significantly increased the activity of ENaC in principal cells in PC-specific GsD mice compared to untreated knock-in mice and CNO treated littermate controls. The cell permeable cAMP analog, 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate, significantly increased the activity and expression in the plasma membrane of recombinant ENaC expressed in CHO and COS-7 cells, respectively. Treatment of PC-specific GsD mice with CNO rapidly and significantly decreased urinary Na
+
excretion compared to untreated PC-specific GsD mice and treated littermate controls. This decrease in Na
+
excretion in response to CNO in PC-specific GsD mice was similar in magnitude and timing as that induced by the selective vasopressin receptor 2 agonist, desmopressin, in wild type mice. These findings demonstrate for the first time that targeted activation of Gs signaling exclusively in PCs is sufficient to increase ENaC activity and decrease dependent urinary Na
+
excretion in live animals.
Transformation of fibroblasts by oncogenic Src causes disruption of actin stress fibers and formation of invasive adhesions called podosomes. Because the small GTPase Rho stimulates stress fiber ...formation, Rho inactivation by Src has been thought to be necessary for stress fiber disruption. However, we show here that RhoGTP levels do not decrease after transformation by activated Src. Inactivation of Rho in Src-transformed fibroblasts by dominant negative RhoA or the Rho-specific inhibitor C3 exoenzyme disrupted podosome structure as judged by localization of podosome components F-actin, cortactin, and Fish. Inhibition of Rho strongly inhibited Src-induced proteolytic degradation of the extracellular matrix. Furthermore, development of an in situ RhoGTP affinity assay allowed us to detect endogenous RhoGTP at podosomes, where it colocalized with F-actin, cortactin, and Fish. Therefore, Rho is not globally inactivated in Src-transformed fibroblasts, but is necessary for the assembly and function of structures implicated in tumor cell invasion.
Abstract Objective Insulin resistance causes type 2 diabetes mellitus and hyperglycemia due to excessive hepatic glucose production and inadequate peripheral glucose uptake. Our objectives were to ...test the hypothesis that the proposed CREB/CRTC2 inhibitor salt inducible kinase 1 (SIK1) contributes to whole body glucose homeostasis in vivo by regulating hepatic transcription of gluconeogenic genes and also to identify novel SIK1 actions on glucose metabolism. Methods We created conditional (floxed) SIK1-knockout mice and studied glucose metabolism in animals with global, liver, adipose or skeletal muscle Sik1 deletion. We examined cAMP-dependent regulation of SIK1 and the consequences of SIK1 depletion on primary mouse hepatocytes. We probed metabolic phenotypes in tissue-specific SIK1 knockout mice fed high fat diet through hyperinsulinemic-euglycemic clamps and biochemical analysis of insulin signaling. Results SIK1 knockout mice are viable and largely normoglycemic on chow diet. On high fat diet, global SIK1 knockout animals are strikingly protected from glucose intolerance, with both increased plasma insulin and enhanced peripheral insulin sensitivity. Surprisingly, liver SIK1 is not required for regulation of CRTC2 and gluconeogenesis, despite contributions of SIK1 to hepatocyte CRTC2 and gluconeogenesis regulation ex vivo. Sik1 mRNA accumulates in skeletal muscle of obese high fat diet-fed mice, and knockout of SIK1 in skeletal muscle, but not liver or adipose tissue, improves insulin sensitivity and muscle glucose uptake on high fat diet. Conclusions SIK1 is dispensable for glycemic control on chow diet. SIK1 promotes insulin resistance on high fat diet by a cell-autonomous mechanism in skeletal muscle. Our study establishes SIK1 as a promising therapeutic target to improve skeletal muscle insulin sensitivity in obese individuals without deleterious effects on hepatic glucose production.
Here, we evaluate the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). We find that lysosomes promote the growth of ...neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. We attribute this paradox to anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. Our findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment.
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•Fly models of lysosomal storage diseases (LSDs) exhibit diminished synaptic growth•Lysosomal protein degradation and MTORC1 activation promote synaptic growth via JNK•MTORC1 phosphorylates Wallenda/DLK1, an MAPKKK upstream of JNK in flies and mammals•ALK inhibition along with high protein diet restores synaptic growth in LSD models
Wong et al. identify an MTORC1-JNK signaling axis in Drosophila and mouse neurons required for synaptic development, which is attenuated upon endolysosomal dysfunction. The authors’ findings suggest a possible therapeutic strategy that may be beneficial for tackling the neurodevelopmental defects commonly observed in lysosomal storage diseases with CNS involvement.
The cAMP‐activated transcription factor CREB stimulates proliferation of myogenic progenitor cells during embryonic development, and genetic activation of CREB stimulates proliferation during muscle ...regeneration. Proliferation of muscle progenitor cells, known as satellite cells, declines during aging. We hypothesized that pharmacologic activation of the cAMP‐CREB pathway would promote satellite cell proliferation after injury and that cAMP‐regulated CREB co‐activators (CRTCs) contribute to CREB‐regulated target gene expression during satellite cell proliferation. To test this, we created ROSA26‐GsDREADD knock‐in mice for tissue‐specific expression of a Gs‐coupled DREADD (Designer Receptor Exclusively Activated by Designer Drugs, “GsD”). After tamoxifen‐induced expression of GsD in muscle satellite cells in young adult mice, activation of the receptor by the GsD ligand CNO (clozapine N‐oxide) increased proliferation after muscle injury. Consistent with our hypothesis, myoblasts over‐expressing constitutively active CRTC2 had increased cell proliferation in vitro, and Crtc2 knockout mice had reduced proliferation after muscle injury. Our results indicate that cAMP signaling enhances satellite cell proliferation in vivo and cAMP regulated CREB coactivator 2 (CRTC2) contributes to the proliferative response after muscle injury. ROSA26‐GsDREADD mice can therefore be used to stimulate cAMP signaling in specific cell populations in vivo to study the effects of pharmacologic activation of cAMP signaling without using ligands that have pleiotropic effects in other cell types.
Support or Funding Information
Research reported in this was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01‐AR059847. DA was supported by a Postdoctoral Fellowship from the American Heart Association (15POST25090134). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Dual Role of SnoN in Mammalian Tumorigenesis Zhu, Qingwei; Krakowski, Ariel R.; Dunham, Elizabeth E. ...
Molecular and Cellular Biology,
01/2007, Letnik:
27, Številka:
1
Journal Article
Under fasting conditions, the cAMP-responsive CREB coactivator TORC2 promotes glucose homeostasis by stimulating the gluconeogenic program in liver. Following its nuclear translocation in response to ...elevations in circulating glucagon, TORC2 regulates hepatic gene expression via an association with CREB on relevant promoters. Here, we show that, in parallel with their effects on glucose output, CREB and TORC2 also enhance insulin signaling in liver by stimulating expression of the insulin receptor substrate 2 (IRS2) gene. The induction of hepatic IRS2 during fasting appears critical for glucose homeostasis; knockdown of hepatic IRS2 expression leads to glucose intolerance, whereas hepatic IRS2 overexpression attenuates the gluconeogenic program and reduces fasting glucose levels. By stimulating the expression of IRS2 in conjunction with gluconeogenic genes, the CREB:TORC2 pathway thus triggers a feedback response that limits glucose output from the liver during fasting.
Manipulation of adrenergic signaling has been shown experimentally and clinically to affect hair follicle growth. In this study, we provide direct evidence that canonical cAMP/CRE-binding protein ...signaling through adrenergic receptors can regulate hair follicle stem cell (HFSC) activation and hair cycle. We found that CRE-binding protein activation is regulated through the hair cycle and coincides with HFSC activation. Both isoproterenol and procaterol, agonists of adrenergic receptors, show the capacity to activate the hair cycle in mice. Furthermore, deletion of ADRB2 receptor, which is thought to mediate sympathetic nervous system regulation of HFSCs, was sufficient to block HFSC activation. Downstream, stimulation of adenylyl cyclase with forskolin or inhibition of phosphodiesterase to increase cAMP accumulation or direct application of cAMP was each sufficient to promote HFSC activation and accelerate initiation of hair cycle. Genetic induction of a Designer Receptors Exclusively Activated by Designer Drug allele showed that G-protein coupled receptor/GαS stimulation, specifically in HFSCs, promoted the activation of the hair cycle. Finally, we provide evidence that G-protein coupled receptor/CRE-binding protein signaling can potentially act on HFSCs by promoting glycolytic metabolism, which was previously shown to stimulate HFSC activation. Together, these data provide mechanistic insights into the role of sympathetic innervation on HFSC function.
During physical exercise, increases in motor neuron activity stimulate the expression of muscle-specific genes through the myocyte enhancer factor 2 (MEF2) family of transcription factors. Elevations ...in intracellular calcium increase MEF2 activity via the phosphorylation-dependent inactivation of class II histone deacetylases (HDACs). In studies to determine the role of the cAMP responsive element binding protein (CREB) in skeletal muscle, we found that mice expressing a dominant-negative CREB transgene (M-ACREB mice) exhibited a dystrophic phenotype along with reduced MEF2 activity. Class II HDAC phosphorylation was decreased in M-ACREB myofibers due to a reduction in amounts of Snf1lk (encoding salt inducible kinase, SIK1), a CREB target gene that functions as a class II HDAC kinase. Inhibiting class II HDAC activity either by viral expression of Snf1lk or by the administration of a small molecule antagonist improved the dystrophic phenotype in M-ACREB mice, pointing to an important role for the SIK1-HDAC pathway in regulating muscle function.
Infections disturb metabolic homeostasis in many contexts, but the underlying connections are not completely understood. To address this, we use paired genetic and computational screens in Drosophila ...to identify transcriptional regulators of immunity and pathology and their associated target genes and physiologies. We show that Mef2 is required in the fat body for anabolic function and the immune response. Using genetic and biochemical approaches, we find that MEF2 is phosphorylated at a conserved site in healthy flies and promotes expression of lipogenic and glycogenic enzymes. Upon infection, this phosphorylation is lost, and the activity of MEF2 changes—MEF2 now associates with the TATA binding protein to bind a distinct TATA box sequence and promote antimicrobial peptide expression. The loss of phosphorylated MEF2 contributes to loss of anabolic enzyme expression in Gram-negative bacterial infection. MEF2 is thus a critical transcriptional switch in the adult fat body between metabolism and immunity.
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•Mef2 is required in Drosophila for immune function and storage of fat and glycogen•MEF2 is phosphorylated in vivo at a conserved site (T20) to promote anabolism•Infection reduces phospho-T20, allowing MEF2 to bind TBP and an immune TATA box•MEF2 dephosphorylation leads to metabolic dysfunction in Gram-negative infection
The transcription factor MEF2 acts as a central switch between metabolic and immune functions in Drosophila, shifting upon infection from activation of anabolic enzymes to immune response genes.
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