Esther Latres

During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.

The human F box protein β-Trcp associates with the Cul1/Skp1 complex and regulates the stability of β-catenin

Ubiquitin-conjugation targets numerous cellular regulators for proteasome-mediated degradation. Thus, the identification of ubiquitin ligases and their physiological substrates is crucially important, especially for those cases in which aberrant levels of regulatory proteins (e.g., β-catenin, p27) result from a deregulated ubiquitination pathway. In yeast, the proteolysis of several G1 regulators is controlled by ubiquitin ligases (or SCFs) formed by three subunits: Skp1, Cul A (Cdc53), and one of many F-box proteins. Specific F-box proteins (Fbps) recruit different substrates to the SCF. Although many Fbps have been identified in mammals, their specific substrates and the existence of multiple SCFs have not yet been reported. We have found that one human Fbp, β-Trcp (β-Transducin repeat containing protein), does indeed form a novel SCF with human Skp1 and Cul1. Consistent with recent reports indicating that Xenopus and Drosophila β-Trcp homologs act as negative regulators of the Wnt/β-catenin signaling pathway, we report here that human β-Trcp interacts with β-catenin in vivo. Furthermore, β-catenin is specifically stabilized in vivo by the expression of a dominant negative β-Trcp. These results indicate that the Cul1/Skp1/β-Trcp complex forms a ubiquitin ligase that mediates the degradation of β-catenin.

Role of the F-box protein Skp2 in lymphomagenesis

The F-box protein Skp2 (S-phase kinase-associated protein 2) positively regulates the G1-S transition by controlling the stability of several G1 regulators, such as the cell cycle inhibitor p27. We show here that Skp2 expression correlates directly with grade of malignancy and inversely with p27 levels in human lymphomas. To directly evaluate the potential of Skp2 to deregulate growth in vivo, we generated transgenic mice expressing Skp2 targeted to the T-lymphoid lineage as well as double transgenic mice coexpressing Skp2 and activated N-Ras. A strong cooperative effect between these two transgenes induced T cell lymphomas with shorter latency and higher penetrance, leading to significantly decreased survival when compared with control and single transgenic animals. Furthermore, lymphomas of Nras single transgenic animals often expressed higher levels of endogenous Skp2 than tumors of double transgenic mice. This study provides evidence of a role for an F-box protein in oncogenesis and establishes SKP2 as a protooncogene causally involved in the pathogenesis of lymphomas.

Limited overlapping roles of P15INK4b and P18INK4c cell cycle inhibitors in proliferation and tumorigenesis

Entry of quiescent cells into the cell cycle is driven by the cyclin D‐dependent kinases Cdk4 and Cdk6. These kinases are negatively regulated by the INK4 cell cycle inhibitors. We report the generation of mice defective in P15INK4b and P18INK4c. Ablation of these genes, either alone or in combination, does not abrogate cell contact inhibition or senescence of mouse embryo fibroblasts in culture. However, loss of P15INK4b, but not of P18INK4c, confers proliferative advantage to these cells and makes them more sensitive to transformation by H‐ras oncogenes. In vivo, ablation of P15INK4b and P18INK4c genes results in lymphoproliferative disorders and tumor formation. Mice lacking P18INK4c have deregulated epithelial cell growth leading to the formation of cysts, mostly in the cortical region of the kidneys and the mammary epithelium. Loss of both P15INK4b and P18INK4c does not result in significantly distinct phenotypic manifestations except for the appearance of cysts in additional tissues. These results indicate that P15INK4b and P18IKN4c are tumor suppressor proteins that act in different cellular lineages and/or pathways with limited compensatory roles.

Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d).

ABSTRACT Male mice lacking both the Ink4c and Ink4dgenes, which encode two inhibitors of D-type cyclin-dependent kinases (Cdks), are infertile, whereas female fecundity is unaffected. Both p18Ink4c and p19Ink4d are expressed in the seminiferous tubules of postnatal wild-type mice, being largely confined to postmitotic spermatocytes undergoing meiosis. Their combined loss is associated with the delayed exit of spermatogonia from the mitotic cell cycle, leading to the retarded appearance of meiotic cells that do not properly differentiate and instead undergo apoptosis at an increased frequency. As a result, mice lacking bothInk4c and Ink4d produce few mature sperm, and the residual spermatozoa have reduced motility and decreased viability. Whether or not Ink4d is present, animals lackingInk4c develop hyperplasia of interstitial testicular Leydig cells, which produce reduced levels of testosterone. The anterior pituitary of fertile mice lacking Ink4c or infertile mice doubly deficient for Ink4c and Ink4d produces normal levels of luteinizing hormone (LH). Therefore, the failure of Leydig cells to produce testosterone is not secondary to defects in LH production, and reduced testosterone levels do not account for infertility in the doubly deficient strain. By contrast,Ink4d-null or double-null mice produce elevated levels of follicle-stimulating hormone (FSH). Because Ink4d-null mice are fertile, increased FSH production by the anterior pituitary is also unlikely to contribute to the sterility observed inInk4c/Ink4d double-null males. Our data indicate that p18Ink4c and p19Ink4d are essential for male fertility. These two Cdk inhibitors collaborate in regulating spermatogenesis, helping to ensure mitotic exit and the normal meiotic maturation of spermatocytes.

Role of F-Box Protein βTrcp1 in Mammary Gland Development and Tumorigenesis

ABSTRACT The F-box protein βTrcp1 controls the stability of several crucial regulators of proliferation and apoptosis, including certain inhibitors of the NF-κB family of transcription factors. Here we show that mammary glands of βTrcp1−/− female mice display a hypoplastic phenotype, whereas no effects on cell proliferation are observed in other somatic cells. To investigate further the role of βTrcp1 in mammary gland development, we generated transgenic mice expressing human βTrcp1 targeted to epithelial cells under the control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. Compared to controls, MMTV βTrcp1 mammary glands display an increase in lateral ductal branching and extensive arrays of alveolus-like protuberances. The mammary epithelia of MMTV βTrcp1 mice proliferate more and show increased NF-κB DNA binding activity and higher levels of nuclear NF-κB p65/RelA. In addition, 38% of transgenic mice develop tumors, including mammary, ovarian, and uterine carcinomas. The targeting of βTrcp1 to lymphoid organs produces no effects on these tissues. In summary, our results support the notion that βTrcp1 positively controls the proliferation of breast epithelium and indicate that alteration of βTrcp1 function and expression may contribute to malignant behavior of breast tumors, at least in part through NF-κB transactivation.

Genetic Deletion of Trb3, the Mammalian Drosophila tribbles Homolog, Displays Normal Hepatic Insulin Signaling and Glucose Homeostasis

Trb3, a mammalian homolog of Drosophila tribbles, was proposed as a suppressor of Akt activity, predominantly in conditions of fasting and diabetes. Given these prior studies, we sought to determine whether Trb3 plays a major role in modulating hepatic insulin sensitivity. To answer this question, we produced mice in which a lacZ reporter was knocked into the locus containing the gene Trib3, resulting in a Trib3 null animal. Trib3 expression analyses demonstrated that the Trib3 is expressed in liver, adipose tissues, heart, kidney, lung, skin, small intestine, stomach, and denervated, but not normal, skeletal muscle. Trib3−/− mice are essentially identical to their wild-type littermates in overall appearance and body composition. Phenotypic analysis of Trib3−/− mice did not detect any alteration in serum glucose, insulin, or lipid levels; glucose or insulin tolerance; or energy metabolism. Studies in Trib3−/− hepatocytes revealed normal Akt and glycogen synthase kinase- 3β phosphorylation patterns, glycogen levels, and expressions of key regulatory gluconeogenic and glycolytic genes. These data demonstrate that deletion of Trib3 has minimal effect on insulin-induced Akt activation in hepatic tissue, and, as such, they question any nonredundant role for Trb3 in the maintenance of glucose and energy homeostasis in mice.

Myostatin blockade with a fully human monoclonal antibody induces muscle hypertrophy and reverses muscle atrophy in young and aged mice

BackgroundLoss of skeletal muscle mass and function in humans is associated with significant morbidity and mortality. The role of myostatin as a key negative regulator of skeletal muscle mass and function has supported the concept that inactivation of myostatin could be a useful approach for treating muscle wasting diseases.MethodsWe generated a myostatin monoclonal blocking antibody (REGN1033) and characterized its effects in vitro using surface plasmon resonance biacore and cell-based Smad2/3 signaling assays. REGN1033 was tested in mice for the ability to induce skeletal muscle hypertrophy and prevent atrophy induced by immobilization, hindlimb suspension, or dexamethasone. The effect of REGN1033 on exercise training was tested in aged mice. Messenger RNA sequencing, immunohistochemistry, and ex vivo force measurements were performed on skeletal muscle samples from REGN1033-treated mice.ResultsThe human monoclonal antibody REGN1033 is a specific and potent myostatin antagonist. Chronic treatment of mice with REGN1033 increased muscle fiber size, muscle mass, and force production. REGN1033 prevented the loss of muscle mass induced by immobilization, glucocorticoid treatment, or hindlimb unweighting and increased the gain of muscle mass during recovery from pre-existing atrophy. In aged mice, REGN1033 increased muscle mass and strength and improved physical performance during treadmill exercise.ConclusionsWe show that specific myostatin antagonism with the human antibody REGN1033 enhanced muscle mass and function in young and aged mice and had beneficial effects in models of skeletal muscle atrophy.

Conditionals by inversion provide a universal method for the generation of conditional alleles

Significance We describe conditional by inversion (COIN), a new design for conditional alleles that uses an optimized conditional gene trap module (COIN module) inserted into the target gene in an orientation opposite to the gene’s direction of transcription. Activation by Cre recombinase inverts the COIN module, resulting in expression of a reporter and termination of transcription, thereby inactivating the target gene while marking the cells where the conditional event has occurred. Creation of COIN alleles for more than 20 genes showed that it is a robust and universal method—applicable to any gene regardless of exon–intron structure—that overcomes the limitations of previous conditional approaches. Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene’s transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon–intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN’s artificial intron opens up engineering modalities for the generation of multifunctional alleles.

The de-ubiquitinating enzyme Unp interacts with the retinoblastoma protein.

The ubiquitin pathway is involved in the proteolytic turnover of many short-lived cellular regulatory proteins. Since selective degradation of substrates of this system requires the covalent attachment of a polyubiquitin chain to the substrates, degradation could be counteracted by de-ubiquitinating enzymes (or isopeptidases) which selectively remove the polyubiquitin chain. Unp is a human isopeptidase with still poorly understood biological functions. Here, we show that cellular Unp specifically interacts with the retinoblastoma gene product (pRb).