Emilio Casanova

Skeletal Muscle-Specific Ablation of raptor, but Not of rictor, Causes Metabolic Changes and Results in Muscle Dystrophy

Mammalian target of rapamycin (mTOR) is a central controller of cell growth. mTOR assembles into two distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2. Here we show that the mTORC1 component raptor is critical for muscle function and prolonged survival. In contrast, muscles lacking the mTORC2 component rictor are indistinguishable from wild-type controls. Raptor-deficient muscles become progressively dystrophic, are impaired in their oxidative capacity, and contain increased glycogen stores, but they express structural components indicative of oxidative muscle fibers. Biochemical analysis indicates that these changes are probably due to loss of activation of direct downstream targets of mTORC1, downregulation of genes involved in mitochondrial biogenesis, including PGC1alpha, and hyperactivation of PKB/Akt. Finally, we show that activation of PKB/Akt does not require mTORC2. Together, these results demonstrate that muscle mTORC1 has an unexpected role in the regulation of the metabolic properties and that its function is essential for life.

Neuronal migration in the murine rostral migratory stream requires serum response factor

The central nervous system is fundamentally dependent on guided cell migration, both during development and in adulthood. We report an absolute requirement of the transcription factor serum response factor (SRF) for neuronal migration in the mouse forebrain. Conditional, late-prenatal deletion of Srf causes neurons to accumulate ectopically at the subventricular zone (SVZ), a prime neurogenic region in the brain. SRF-deficient cells of the SVZ exhibit impaired tangential chain migration along the rostral migratory stream into the olfactory bulb. SVZ explants display retarded chain migration in vitro. Regarding target genes, SRF deficiency impairs expression of the β-actin and gelsolin genes, accompanied by reduced cytoskeletal actin fiber density. At the posttranslational level, cofilin, a key regulator of actin dynamics, displays dramatically elevated inhibitory phosphorylation at Ser-3. Our studies indicate that SRF-controlled gene expression directs both the structure and dynamics of the actin microfilament, thereby determining cell-autonomous neuronal migration.

A novel Ncr1-Cre mouse reveals the essential role of STAT5 for NK-cell survival and development.

We generated a transgenic mouse line that expresses the Cre recombinase under the control of the Ncr1 (p46) promoter. Cre-mediated recombination was tightly restricted to natural killer (NK) cells, as revealed by crossing Ncr1-iCreTg mice to the eGFP-LSLTg reporter strain. Ncr1-iCreTg mice were further used to study NK cell-specific functions of Stat5 (signal transducers and activators of transcription 5) by generating Stat5(f/f) Ncr1-iCreTg animals. Stat5(f/f) Ncr1-iCreTg mice were largely devoid of NK cells in peripheral lymphoid organs. In the bone marrow, NK-cell maturation was abrogated at the NK cell-precursor stage. Moreover, we found that in vitro deletion of Stat5 in interleukin 2-expanded NK cells was incompatible with NK-cell viability. In vivo assays confirmed the complete abrogation of NK cell-mediated tumor control against B16F10-melanoma cells. In contrast, T cell-mediated tumor surveillance against MC38-adenocarcinoma cells was undisturbed. In summary, the results of our study show that STAT5 has a cell-intrinsic role in NK-cell development and that Ncr1-iCreTg mice are a powerful novel tool with which to study NK-cell development, biology, and function.

Stat3 Is a Negative Regulator of Intestinal Tumor Progression in ApcMin Mice

BACKGROUND AND AIMS The transcription factor signal transducer and activator of transcription 3 (Stat3) has been considered to promote progression and metastasis of intestinal cancers. METHODS We investigated the role of Stat3 in intestinal tumors using mice with conditional ablation of Stat3 in intestinal epithelial cells (Stat3(DeltaIEC)). RESULTS In the Apc(Min) mouse model of intestinal cancer, genetic ablation of Stat3 reduced the multiplicity of early adenomas. However, loss of Stat3 promoted tumor progression at later stages, leading to formation of invasive carcinomas, which significantly shortened the lifespan of Stat3(DeltaIEC)Apc(Min/+) mice. Interestingly, loss of Stat3 in tumors of Apc(Min/+) mice had no significant impact on cell survival and angiogenesis, but promoted cell proliferation. A genome-wide expression analysis of Stat3-deficient tumors suggested that Stat3 might negatively regulate intestinal cancer progression via the cell adhesion molecule CEACAM1. CONCLUSIONS Our data suggest that Stat3 impairs invasiveness of intestinal tumors. Therefore, therapeutic targeting of the Stat3 signaling pathway in intestinal cancer should be evaluated for adverse effects on tumor progression.

NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1

GABAB receptors are the G-protein–coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GABAB receptors are abundant on dendritic spines, where they dampen postsynaptic excitability and inhibit Ca2+ influx through NMDA receptors when activated by spillover of GABA from neighboring GABAergic terminals. Here, we show that an excitatory signaling cascade enables spines to counteract this GABAB-mediated inhibition. We found that NMDA application to cultured hippocampal neurons promotes dynamin-dependent endocytosis of GABAB receptors. NMDA-dependent internalization of GABAB receptors requires activation of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), which associates with GABAB receptors in vivo and phosphorylates serine 867 (S867) in the intracellular C terminus of the GABAB1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABAB receptors refractory to NMDA-mediated internalization. Time-lapse two-photon imaging of organotypic hippocampal slices reveals that activation of NMDA receptors removes GABAB receptors within minutes from the surface of dendritic spines and shafts. NMDA-dependent S867 phosphorylation and internalization is predominantly detectable with the GABAB1b subunit isoform, which is the isoform that clusters with inhibitory effector K+ channels in the spines. Consistent with this, NMDA receptor activation in neurons impairs the ability of GABAB receptors to activate K+ channels. Thus, our data support that NMDA receptor activity endocytoses postsynaptic GABAB receptors through CaMKII-mediated phosphorylation of S867. This provides a means to spare NMDA receptors at individual glutamatergic synapses from reciprocal inhibition through GABAB receptors.

Impairment of hepatic growth hormone and glucocorticoid receptor signaling causes steatosis and hepatocellular carcinoma in mice

Growth hormone (GH)‐activated signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid (GC)‐responsive glucocorticoid receptor (GR) are important signal integrators in the liver during metabolic and physiologic stress. Their deregulation has been implicated in the development of metabolic liver diseases, such as steatosis and progression to fibrosis. Using liver‐specific STAT5 and GR knockout mice, we addressed their role in metabolism and liver cancer onset. STAT5 single and STAT5/GR double mutants developed steatosis, but only double‐mutant mice progressed to liver cancer. Mechanistically, STAT5 deficiency led to the up‐regulation of prolipogenic sterol regulatory element binding protein 1 (SREBP‐1) and peroxisome proliferator activated receptor gamma (PPAR‐γ) signaling. Combined loss of STAT5/GR resulted in GH resistance and hypercortisolism. The combination of both induced expression of adipose tissue lipases, adipose tissue lipid mobilization, and lipid flux to the liver, thereby aggravating STAT5‐dependent steatosis. The metabolic dysfunctions in STAT5/GR compound knockout animals led to the development of hepatic dysplasia at 9 months of age. At 12 months, 35% of STAT5/GR‐deficient livers harbored dysplastic nodules and ∼60% hepatocellular carcinomas (HCCs). HCC development was associated with GH and insulin resistance, enhanced tumor necrosis factor alpha (TNF‐α) expression, high reactive oxygen species levels, and augmented liver and DNA damage parameters. Moreover, activation of the c‐Jun N‐terminal kinase 1 (JNK1) and STAT3 was prominent. Conclusion: Hepatic STAT5/GR signaling is crucial for the maintenance of systemic lipid homeostasis. Impairment of both signaling cascades causes severe metabolic liver disease and promotes spontaneous hepatic tumorigenesis. (HEPATOLOGY 2011;54:1398–1409)

No evidence for a bone phenotype in GPRC6A knockout mice under normal physiological conditions

GPRC6A is a seven-transmembrane receptor mediating signaling by a wide range of L-alpha-amino acids, a signaling augmented by the divalent cations Ca(2)(+) and Mg(2)(+). GPRC6A transcripts are detected in numerous mammalian tissues, but the physiological role of the receptor is thus far elusive. Analogously to the closely related calcium-sensing receptor, GPRC6A has been proposed to function as a metabolic sensor of Ca(2)(+) and amino acids in bone and other tissues. In the present study, we have generated the first GPRC6A knockout mice and studied their phenotype with particular focus on bone homeostasis. The generated GPRC6A knockout mice are viable and fertile, develop normally, and exhibit no significant differences in body weight compared with wild-type littermates. Assessment of bone mineral density, histomorphometry, and bone metabolism demonstrated no significant differences between 13-week-old knockout and wild-type mice. In conclusion, our data do not support a role for GPRC6A in normal bone physiology.

Expression of Cre recombinase in dopaminoceptive neurons

BackgroundDopamine-activated signaling regulates locomotor and emotional responses and alterations in dopamine-signaling are responsible of several psychomotor disorders. In order to identify specific functions of these pathways, the Cre/loxP system has been used. Here, we describe the generation and the characterization of a transgenic mouse line expressing the Cre recombinase in dopaminoceptive neurons. To this purpose, we used as expression vector a 140 kb yeast artificial chromosome (YAC) containing the dopamine D1 receptor gene (Drd1a).ResultsIn the chosen line, D1Cre, the spatio-temporal pattern of Cre expression closely recapitulated that of the endogenous Drd1a gene, as assessed by immunohistological approaches in embryonic and adult stages. Efficiency of recombination was confirmed by crossing D1Cre with three different loxP lines (Creb1loxP, CaMKIVloxP and GRloxP) and with the R26R reporter. In the three loxP lines studied, recombination was restricted to the area of Cre expression.ConclusionIn view of the patterns of recombination restricted to the major dopaminoceptive regions as seen in the context of the CREB, CaMKIV and GR mutations, the D1Cre line will be a useful tool to dissect the contributions of specific genes to biological processes involving dopamine signaling.

Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis

STAT3 is considered to play an oncogenic role in several malignancies including lung cancer; consequently, targeting STAT3 is currently proposed as therapeutic intervention. Here we demonstrate that STAT3 plays an unexpected tumour-suppressive role in KRAS mutant lung adenocarcinoma (AC). Indeed, lung tissue-specific inactivation of Stat3 in mice results in increased KrasG12D-driven AC initiation and malignant progression leading to markedly reduced survival. Knockdown of STAT3 in xenografted human AC cells increases tumour growth. Clinically, low STAT3 expression levels correlate with poor survival and advanced malignancy in human lung AC patients with smoking history, which are prone to KRAS mutations. Consistently, KRAS mutant lung tumours exhibit reduced STAT3 levels. Mechanistically, we demonstrate that STAT3 controls NF-κB-induced IL-8 expression by sequestering NF-κB within the cytoplasm, thereby inhibiting IL-8-mediated myeloid tumour infiltration and tumour vascularization and hence tumour progression. These results elucidate a novel STAT3–NF-κB–IL-8 axis in KRAS mutant AC with therapeutic and prognostic relevance.

When reverse genetics meets physiology: the use of site‐specific recombinases in mice

The use of site‐specific recombinases enables the precise introduction of defined genetic mutations into the mouse genome. In theory, any deletion, point mutation, inversion or translocation can be modeled in mice. Because gene targeting is controlled both spatially and temporally, the function of a given gene can be studied in the desired cell types and at a specific time point. This ‘genetic dissection’ allows to define gene function in development, physiology or behavior. In this review, we focus on the technical possibilities of Cre and other site‐specific recombinases but also discuss their limitations.