Nadia Messaddeq

Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation

While bile acids (BAs) have long been known to be essential in dietary lipid absorption and cholesterol catabolism, in recent years an important role for BAs as signalling molecules has emerged. BAs activate mitogen-activated protein kinase pathways, are ligands for the G-protein-coupled receptor (GPCR) TGR5 and activate nuclear hormone receptors such as farnesoid X receptor α (FXR-α; NR1H4). FXR-α regulates the enterohepatic recycling and biosynthesis of BAs by controlling the expression of genes such as the short heterodimer partner (SHP; NR0B2) that inhibits the activity of other nuclear receptors. The FXR-α-mediated SHP induction also underlies the downregulation of the hepatic fatty acid and triglyceride biosynthesis and very-low-density lipoprotein production mediated by sterol-regulatory-element-binding protein 1c. This indicates that BAs might be able to function beyond the control of BA homeostasis as general metabolic integrators. Here we show that the administration of BAs to mice increases energy expenditure in brown adipose tissue, preventing obesity and resistance to insulin. This novel metabolic effect of BAs is critically dependent on induction of the cyclic-AMP-dependent thyroid hormone activating enzyme type 2 iodothyronine deiodinase (D2) because it is lost in D2-/- mice. Treatment of brown adipocytes and human skeletal myocytes with BA increases D2 activity and oxygen consumption. These effects are independent of FXR-α, and instead are mediated by increased cAMP production that stems from the binding of BAs with the G-protein-coupled receptor TGR5. In both rodents and humans, the most thermogenically important tissues are specifically targeted by this mechanism because they coexpress D2 and TGR5. The BA–TGR5–cAMP–D2 signalling pathway is therefore a crucial mechanism for fine-tuning energy homeostasis that can be targeted to improve metabolic control.

Essential role of α6 integrins in cortical and retinal lamination

Extracellular matrix (ECM) is believed to play important roles in many aspects of nervous system development [1]. The laminins are ECM glycoproteins expressed in neural tissues and are potent stimulators of neurite outgrowth in vitro [1-3]. Genetic approaches using Drosophila and Caenorhabditis elegans have demonstrated a role for laminin and a laminin receptor in vivo in axon pathfinding and fasciculation, respectively [4,5]. In higher organisms, however, the role of laminins in the development of the nervous system is poorly understood. Integrins alpha 6 beta 1 and alpha 6 beta 4 are major laminin receptors. A role for the alpha 6 integrin in neurulation has been reported in amphibians [6]. We previously described mice lacking integrin alpha 6; these mice died at birth with severe skin blistering [7]. Detailed analyses of integrin alpha 6-/- mice reported here revealed abnormalities in the laminar organization of the developing cerebral cortex and retina. Ectopic neuroblastic outgrowths were found on the brain surface and in the vitreous body in the eye. Alterations of laminin deposition were found in mutant brains. Thus, this study provides evidence for an essential role of integrin-laminin interactions in the proper development of the nervous system. These observations are particularly significant given the recent report that human patients suffering from epidermolysis bullosa can carry mutations in ITGA6, the gene encoding the alpha 6 integrin chain [8,9].

Oral-facial-digital type I protein is required for primary cilia formation and left-right axis specification.

The oral-facial-digital type I (OFD1) syndrome (OMIM 311200) is a human developmental disorder; affected individuals have craniofacial and digital abnormalities and, in 15% of cases, polycystic kidney. The disease is inherited as an X-linked dominant male-lethal trait. Using a Cre-loxP system, we generated knockout animals lacking Ofd1 and reproduced the main features of the disease, albeit with increased severity, possibly owing to differences of X inactivation patterns between human and mouse. We found failure of left-right axis specification in mutant male embryos, and ultrastructural analysis showed a lack of cilia in the embryonic node. Formation of cilia was defective in cystic kidneys from heterozygous females, implicating ciliogenesis as a mechanism underlying cyst development. In addition, we found impaired patterning of the neural tube and altered expression of the 5′ Hoxa and Hoxd genes in the limb buds of mice lacking Ofd1, suggesting that Ofd1 could have a role beyond primary cilium organization and assembly.

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate–binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.

The lipid phosphatase myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice

Myotubularin is a ubiquitously expressed phosphatase that acts on phosphatidylinositol 3-monophosphate [PI(3)P], a lipid implicated in intracellular vesicle trafficking and autophagy. It is encoded by the MTM1 gene, which is mutated in X-linked myotubular myopathy (XLMTM), a muscular disorder characterized by generalized hypotonia and muscle weakness at birth leading to early death of most affected males. The disease was proposed to result from an arrest in myogenesis, as the skeletal muscle from patients contains hypotrophic fibers with centrally located nuclei that resemble fetal myotubes. To understand the physiopathological mechanism of XLMTM, we have generated mice lacking myotubularin by homologous recombination. These mice are viable, but their lifespan is severely reduced. They develop a generalized and progressive myopathy starting at around 4 weeks of age, with amyotrophy and accumulation of central nuclei in skeletal muscle fibers leading to death at 6–14 weeks. Contrary to expectations, we show that muscle differentiation in knockout mice occurs normally. We provide evidence that fibers with centralized myonuclei originate mainly from a structural maintenance defect affecting myotubularin-deficient muscle rather than a regenerative process. In addition, we demonstrate, through a conditional gene-targeting approach, that skeletal muscle is the primary target of murine XLMTM pathology. These mutant mice represent animal models for the human disease and will be a valuable tool for understanding the physiological role of myotubularin.

Developmental expression pattern of Stra6, a retinoic acid-responsive gene encoding a new type of membrane protein

Retinoic acid plays important roles in development, growth and differentiation by regulating the expression of target genes. A new retinoic acid-inducible gene, Stra6, has been identified in P19 embryonal carcinoma cells using a subtractive hybridization cDNA cloning technique. Stra6 codes for a very hydrophobic membrane protein of a new type, which does not display similarities with previously characterized integral membrane proteins. Stra6, which exhibits a specific pattern of expression during development and in the adult, is strongly expressed at the level of blood-organ barriers. Interestingly, in testis Sertoli cells, Stra6 has a spermatogenic cycle-dependent expression which is lost in testes of RAR alpha null mutants where Stra6 is expressed in all tubules. We suggest that the Stra6 protein may be a component of an as yet unidentified transport machinery.

Liver receptor homolog 1 is essential for ovulation

Female fertility requires normal ovarian follicular growth and ovulation. The nuclear receptor liver receptor homolog 1 has been implicated in processes as diverse as bile acid metabolism, steroidogenesis, and cell proliferation. In the ovary, Lrh1 is expressed exclusively in granulosa and luteal cells. Using somatic targeted mutagenesis, we show that mice lacking Lrh1 in granulosa cells are sterile, due to anovulation. The preovulatory stimulus fails to elicit cumulus expansion, luteinization, and follicular rupture in these mice. Multiple defects, including severely reduced transactivation of the Lrh1 target gene, nitric oxide synthase 3, leads to increased intrafollicular estradiol levels in the absence of Lrh1. This further causes dysfunction of prostaglandin and hyaluronic acid cascades and interrupts cumulus expansion. Lack of Lrh1 also interferes with progesterone synthesis because of failure of normal expression of the Lrh1 targets, steroidogenic acute regulatory protein and cytochrome P450 side-chain cleavage. In addition, expression of extracellular matrix proteases essential for ovulation is compromised. These results demonstrate that Lrh1 is a regulator of multiple mechanisms essential for maturation of ovarian follicles and for ovulation. Lrh1 is therefore a key modulator of female fertility and a potential target for contraception.

Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a target of 5-HT2B-receptor signaling

Identification of factors regulating cardiomyocyte survival and growth is important to understand the pathogenesis of congenital heart diseases. Little is known about the molecular mechanism of cardiac functions triggered by serotonin. The link between signaling circuitry of external stimuli and the mitochondrial apoptotic machinery is of wide interest in cardiac diseases. Using cultured cardiomyocytes and 5‐hydroxytryptamine (5‐HT)2B‐receptor knockout mice as an animal model of dilated cardiomyopathy, for the first time we show that serotonin via the Gq‐coupled 5‐HT2B‐receptor protect cardiomyocytes against serum deprivation‐induced apoptosis as manifested by DNA fragmentation, nuclear chromatin condensation, and TUNEL labeling. Serotonin prevents cytochrome c release and caspase‐9 and ‐3 activation after serum deprivation via cross‐talks between phosphatidylinositol‐3 kinase/Akt and extracellular signal‐regulated kinase (ERK) 1/2 signaling pathways. Serotonin binding to 5‐HT2B‐receptor activates ERK kinases to inhibit Bax expression induced by serum deprivation. Serotonin via phosphatidylinositol‐3 kinase/Akt can activate NF‐κB that is required for the regulation of the mitochondrial adenine nucleotide translocator (ANT‐1). Parallel to these observations, ultrastructural analysis in the 5‐HT2B‐receptor knockout mice heart revealed pronounced mitochondrial defects in addition to altered mitochondrial enzyme activities (cytochrome oxidase and succinate dehydrogenase) and ANT‐1 and Bax expressions. These findings identify 5‐HT as a novel survival factor targeting mitochondria in cardiomyocytes.

Physiological and retinoid-induced proliferations of epidermis basal keratinocytes are differently controlled.

To investigate the roles of retinoic acid (RA) receptors (RARs) in the physiology of epidermis that does not express RARβ, conditional spatio‐temporally controlled somatic mutagenesis was used to selectively ablate RARα in keratinocytes of RARγ‐null mice. Keratinocyte proliferation was maintained in adult mouse epidermis lacking both RARα and RARγ, as well as in RARβ‐null mice. All RAR‐mediated signalling pathways are therefore dispensable in epidermis for homeostatic keratinocyte renewal. However, topical treatment of mouse skin with selective retinoids indicated that RXR/RARγ heterodimers, in which RXR transcriptional activity was subordinated to that of its RARγ partner, were required for retinoid‐induced epidermal hyperplasia, whereas RXR homodimers and RXR/RARα heterodimers were not involved. RA‐induced keratinocyte proliferation was studied in mutant mice in which RXRα, RXRα and RARα, RARγ, or RXRα and RARγ genes were specifically disrupted in either basal or suprabasal keratinocytes. We demonstrate that the topical retinoid signal is transduced by RXRα/RARγ heterodimers in suprabasal keratinocytes, which, in turn, stimulate proliferation of basal keratinocytes via a paracrine signal that may be heparin‐binding EGF‐like growth factor.

T-tubule disorganization and defective excitation-contraction coupling in muscle fibers lacking myotubularin lipid phosphatase

Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1-deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca2+ release elicited by voltage-clamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic Ca2+ removal and SR Ca2+ content essentially unaffected. At the molecular level, Mtm1-deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca2+ release is responsible for the failure of muscle function in myotubular myopathy.