Table of Contents

Volume 7, Issue 14

  July 15, 2008

  Pages 2081 - 2270

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NEWS

Open Access Article

Transposons find a small RIP in NFκB pathway

Julia V. Kichina
Pages 2081 - 2082
http://dx.doi.org/10.4161/cc.7.14.6598
Abstract | PDF

Hyperactivation of NFκB is common in a variety of human diseases. Although numerous transient regulators have been found, it is still unclear which ones can permanently sustain the abnormal status of NFκB in circumvention of several feedback mechanisms and without the associated toxicity. Activation of NFκB could be used to build readouts for genetic screening, and this phenotype has been interrogated using various forward genetics techniques. Mutants with altered activity of NFκB have been obtained using chemical mutagenesis or selected from libraries of full-length cDNAs, genetic suppressor elements or small interfering RNAs. More recently, reversible insertional mutagenesis using retroviral vectors has been tried in this system as well. The authors of the latter report used insertion of a strong promoter to generate dominant mutants, which could be distinguished from the spontaneous mutants by their dependence on the promoter function. In the current issue, the same group describes a similar approach with a new set of tools, based on the modified Sleeping Beauty transposons. Transposon-based insertion of a promoter has been used before for in vivo mutagenesis, but the new study is the first reported use of this strategy in cultured cells. Importantly, the earlier examples of insertional mutagenesis with similar transposons used to produce lists of candidate targets that were chosen among many insertion sites based on certain statistical assumptions or prior knowledge of the nearby genes. In contrast, Dasgupta and colleagues used regulation of the inserted promoter to unambiguously prove the relevance of the targeted locus to the mutant phenotype (Figure 1). The insertional mutant that was characterized in most details revealed expression of the C-terminal fragment of RIP1. The role of RIP1 in signaling to NFκB is well-recognized, yet the finding is unexpected. The N-terminal portion of RIP1 is comprised of a putative kinase domain, although its kinase activity appears dispensable for the activation of NFκB. In the C-terminal portion, the intermediate domain is followed by “RIP homotypic interaction motif” and the DEATH domain, and most confirmed interactors of RIP1 bind to this part of the protein. However, prior reports claimed that a C-terminal fragment produced by caspase cleavage within the intermediate domain of the protein has a dominant-negative effect because in its presence TNFα treatment fails to superinduce an NFκB reporter. Instead, Dasgupta et al. demonstrated that without additional extracellular stimuli a similar short form of RIP1 can activate NFκB and other transcription factors. The state of the cell upon expression of the short RIP1 resembles the consequence of TNFα treatment, including the characteristic dependence on NFκB for survival. However, at the same time the cell is made unresponsive to externally added TNFα because the full-length RIP1, which is the centerpiece of TNFα signaling, is no longer present. This is the most exciting finding of the study: it points to a previously unrecognized mechanism that controls the abundance of RIP1 and, by inference, the responsiveness of the signaling cascades that depend on this molecule. There is now a tantalizing possibility that the same mechanism could be engaged for the therapeutic management of human conditions, such as septic shock, where efficient suppression of TNFα signaling is of vital importance. The presented bioinformatic and experimental datasuggests that a short form of RIP1 may be naturally produced from an internal promoter in the human ripk1 gene. The regulation of this promoter is worth studying further. The longer RIP1 is notoriously toxic, but the shorter variant appears better tolerated. This, and the independence of extracellular stimuli, makes the short RIP1 a more likely candidate for constitutive activation of NFκB in human malignancies. Additional work is needed to confirm this possibility.

Further reading:

1. Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 2003; 3:745-56.
2. Dasgupta M, Agarwal MK, Varley P, Lu T, Stark GR, Kandel ES. Transposon-based mutagenesis identifies short RIP1 as an activator of NF-kB. Cell Cycle 2008; 7; In this issue.
3. Kandel ES, Lu T, Wan Y, Agarwal MK, Jackson MW, Stark GR. Mutagenesis by reversible promoter insertion to study the activation of NF-kappaB. Proc Natl Acad Sci USA 2005; 102:6425-30.
4. Kandel ES, Stark GR. "Forward genetics in mammalian cells." Signal Transducers and Activators of Transcription (STATs): Activation and Biology. Ed., Sehgal PB, Levy DE, Hirano T. The Netherlands, Kluwer Academic Publishers.
5. Meylan E, Tschopp J. The RIP kinases: crucial integrators of cellular stress. Trends Biochem Sci 2005; 30:151-9.
Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 2007; 8:49-62.




EXTRA VIEWS

Open Access Article

Finding an “Achilles’ heel” of cancer: The role of glucose and glutamine metabolism in the survival of transformed cells

Mariia Yuneva
Pages 2083 - 2089
http://dx.doi.org/10.4161/cc.7.14.6256
Abstract | PDF

Multiple lines of evidence indicate that the process of tumorigenesis is often associated with altered metabolism of two major nutrients, glucose and glutamine. These two nutrients are engaged in multiple metabolic pathways that can be required for cell viability. The roles of glucose and glutamine in the survival of transformed cells both in vitro and in vivo have been separately evaluated in various cell systems, and glucose as the major cellular energy source has received most of the attention. At the same time, data suggests that the inclusion of glucose and glutamine into specific metabolic pathways and cellular sensitivity to the availability of either of these nutrients depends on the cell origin and the combination and nature of transforming events. Exploiting cell metabolism to develop selective cancer therapeutics requires consideration of these factors and evaluation of the requirement of glucose and glutamine metabolism for survival of different transformed cells. Here we discuss possible molecular mechanisms underlying oncogene-induced sensitivity to deprivation of these nutrients.

Open Access Article

TWIST activation by hypoxia inducible factor-1 (HIF-1): Implications in metastasis and development

Muh-Hwa Yang and Kou-Juey Wu
Pages 2090 - 2096
http://dx.doi.org/10.4161/cc.7.14.6324
Abstract | PDF

Hypoxia is a microenvironmental factor which plays a critical role in development and tumor progression. The hypoxic response is mainly mediated by hypoxia inducible factor-1 (HIF-1) composed of HIF-1alpha and HIF-1beta, which becomes active under low oxygen condition. HIF-1 activates the transcription of hypoxia inducible genes which regulate diverse cellular functions including metabolism, angiogenesis, and invasion. In cancer metastasis, HIF-1-regulated genes promote angiogenesis, invasion, and epithelial-mesenchymal transition (EMT), a critical step of metastasis. TWIST is a master regulator of gastrulation and mesoderm specification and is recently implicated to be essential to mediate cancer metastasis. We recently showed that HIF-1 promotes EMT through direct regulation of TWIST expression. TWIST is critical for hypoxia mediated EMT and metastasis. TWIST plays a non-redundant role in relation to other EMT regulators (e.g. Snail) under hypoxia. Co-expression of HIF-1alpha, TWIST and Snail could be used as a prognostic marker in cancer patients. These findings suggest that hypoxia and/or HIF-1 orchestrates EMT and metastasis through the coordinated regulation of different EMT regulators. Our results provide the important link between hypoxia and developmental processes regulated by TWIST. The implications of the roles of hypoxia and TWIST in early embryonic development are discussed.

Open Access Article

A wide-ranging cellular response to UV damage of DNA

Matthew Stokes and Michael Comb
Pages 2097 - 2099
http://dx.doi.org/10.4161/cc.7.14.6326
Abstract | PDF

The kinases ATM and ATR are central to proper function of the DNA damage response. These kinases phosphorylate proteins to coordinate cell cycle progression and DNA damage repair/bypass. We have recently reported a large-scale identification of ATM/ATR substrates phosphorylated in response to UV damage of DNA. Overall 231 sites of phosphorylation were induced by UV damage of DNA or dependent on proper function of ATR. The study expanded the number of phosphorylation sites from protein classes known to be involved in the DNA damage response. Further, many sites were identified from protein types not thought to have a role in damage signaling. This observation suggests that the DNA damage response affects a much wider range of cellular processes than was previously appreciated. This study has also extended the successful use of the PhosphoScan® proteomic method from phospho-tyrosine to serine/threonine motifs, providing a general blueprint to use the method to study signaling pathways underlying a wide range of diseases.

Open Access Article

Mitotic regulation of SREBP and ATF6 by separation of the Golgi and ER

René Bartz and Joachim Seemann
Pages 2100 - 2105
http://dx.doi.org/10.4161/cc.7.14.6327
Abstract | PDF

The highly organized structure of the Golgi apparatus becomes compromised during mitosis. The Golgi cisternae unstack and form vesicles, which are then partitioned into the daughter cells. Here, we analyzed the fate of the SREBP and ATF6, two key transcription factors of sterol biogenesis and the unfolded protein response, respectively. During interphase the precursors of both transcription factors are silent in the ER, but upon activation signals SREBP and ATF6 move to the Golgi where they are activated by proteolytic cleavage by S1P and S2P. Since the spatial separation between the ER and the Golgi is the basis for the regulated activation, we investigated whether mitosis is sufficient to trigger activation. We found that the strict compartmentalization of the ER and the Golgi is maintained during mitosis to inhibit the activation of SREBP and ATF6.




PERSPECTIVES

Open Access Article

Targeting intercellular signals for bone regeneration from bone marrow mesenchymal progenitors

Fanxin Long
Pages 2106 - 2111
http://dx.doi.org/10.4161/cc.7.14.6257
Abstract | PDF

There are tremendous unmet clinical needs for effective strategies to enhance bone regeneration in vivo. The sustained presence of multipotent mesenchymal progenitors in the bone marrow in aged and osteoporotic individuals offers the potential for therapeutic interventions to induce osteoblast production from the resident progenitors. Recent advances in understanding the intercellular signals governing osteogenic decisions may provide targets for developing novel bone-enhancing therapeutics.

Open Access Article

Akt and mRNA translation by interferons

Surinder Kaur, Efstratios Katsoulidis and Leonidas C. Platanias
Pages 2112 - 2116
http://dx.doi.org/10.4161/cc.7.14.6258
Abstract | PDF

The important antiviral and antitumor properties of interferons (IFNs) in vitro and in vivo have triggered extensive investigations over the years to understand the signals that control such responses in normal and malignant cells. The discovery of IFN-regulated Jak-Stat pathways and various ancillary cascades has led to the definition and establishment of models by which early signals at the IFN receptor level ultimately induce transcription of IFN-controlled genes to generate antiviral and antitumor responses. An important outstanding issue in the field has been the identification of the mechanisms responsible for regulation of mRNA translation of IFN-sensitive genes. There is emerging evidence suggesting that mTOR and its effectors play key and essential roles in the generation of such responses. Moreover, recent studies point towards Akt as a common and central integrator for such responses in Type I and II IFN signaling, via its regulatory effects on mTOR. Here, we review the accumulating evidence on the importance of Akt in IFN-signaling, with particular emphasis on its role in mRNA translation of IFN-sensitive genes. The implications of such studies on the overall perception of the Akt pathway are also discussed.

Open Access Article

Emerging roles of SIRT1 in vascular endothelial homeostasis

Michael Potente and Stefanie Dimmeler
Pages 2117 - 2122
http://dx.doi.org/10.4161/cc.7.14.6267
Abstract | PDF

Sir2 is a NAD+-dependent deacetylase, which regulates life span in multiple model organisms in response to caloric restriction. Mammalian homologues of Sir2 comprise a family of seven proteins termed sirtuins (SIRT1-SIRT7), which have gained considerable attention for their impact on several important physiological processes associated with metabolism and stress resistance. In addition, recent studies point to SIRT1 as a key regulator of vascular endothelial homeostasis controlling angiogenesis, vascular tone and endothelial dysfunction. Here, we review the emerging role of SIRT1 as an important modulator of signaling networks critical for maintaining vascular endothelial homeostasis and discuss SIRT1 as a potential therapeutic target for cardiovascular diseases in the adult.

Open Access Article

Systems biology of mammalian cell division

Ralf Kittler, Laurence Pelletier and Frank Buchholz
Pages 2123 - 2128
http://dx.doi.org/10.4161/cc.7.14.6322
Abstract | PDF

High-throughput screening technologies allow the identification of genes and proteins essential for mammalian cell division. However, the underlying complexity and connectivity of different biological processes, such as signal transduction, transcription, translation and proteolysis make it difficult to understand the mammalian cell cycle based on analysis of its individual components alone. The recent development of robust and precise assays to study the mammalian cell cycle, in combination with functional genomics and proteomics, together provide the necessary tools to address this critical issue. With the implementation of different “Omics” technologies for quantitative and high-throughput data acquisition, the possibility of obtaining a more detailed view of the mammalian cell cycle is now realistic. Here, we review RNAi reagents, assays and validation strategies for the identification of genes functioning in the human cell cycle, and outline genomic, proteomic and microscopic approaches to further characterize their specific functions. While a fully integrated model of mammalian cell division remains a distant goal, a framework of a systems understanding of this medically relevant process is beginning to emerge.

Open Access Article

Cell cycle regulation in hair cell development and regeneration in the mouse cochlea

Zhiyong Liu and Jian Zuo
Pages 2129 - 2133
http://dx.doi.org/10.4161/cc.7.14.6423
Abstract | PDF

Cell cycle inhibitors play important roles in the development of mammalian cochleae. Loss of function of those factors in mice at various developmental stages results in distinct phenotypes characterized by overproduction or loss of cochlear sensory cells. Our recent study showed that acute deletion of the retinoblastoma protein (Rb) induces rapid cell cycle reentry and subsequent loss of postnatal cochlear hair cells in mice. Clearly, these regulators play multiple roles in cell cycle exit and differentiation of hair cell and supporting cell progenitors. They are also crucial in maintenance of postmitotic states and survival of differentiated hair cells and supporting cells. In mammals, lost hair cells cannot be spontaneously replaced, leading to permanent deafness. However, lower vertebrates such as birds and fish can naturally regenerate damaged hair cells from the underlying supporting cells through proliferation and transdifferentiation. Thus, manipulating cell cycle inhibitors in mammalian cochleae could provide a new avenue to restore hearing in deaf people caused by a variety of genetic mutations and environmental insults.




REVIEW

Open Access Article

Telomere maintenance in Drosophila: Rapid transposon evolution at chromosome ends

Alfredo Villasante, Beatriz de Pablos, María Méndez-Lago and José P. Abad
Pages 2134 - 2138
http://dx.doi.org/10.4161/cc.7.14.6275
Abstract | PDF

The maintenance of terminal sequences is an important role of the telomere, since it prevents the loss of internal regions that encode essential genes. In most eukaryotes, this is accomplished by the telomerase. However, telomere length can also be maintained by other mechanisms, such as homologous recombination and transposition of telomeric retrotransposons to the chromosome ends. A remarkable situation is the case of Drosophila, where telomerase was lost, and thus telomeres managed to be maintained by occasional retrotransposition of telomeric elements to the receding ends. In the recent analysis of 12 Drosophila genomes, ¬¬the multiplicity of autonomous and non-autonomous telomere-specific retrotransposons has revealed extensive and rapid evolution of telomeric DNA. The phylogenetic relationship among these telomeric retrotransposons is congruent with the species phylogeny, suggesting that they have been vertically transmitted from a common ancestor. In this review, we also suggest that the formation of a non-canonical DNA structure at Drosophila telomeres could be the way to protect the ends.




BRIEF REPORT

Open Access Article

A novel effect of DNA methyltransferase and histone deacetylase inhibitors : NFκB inhibition in malignant myeloblasts

Claire Fabre, Jennifer Grosjean, Maximilien Tailler, Simone Boehrer, Lionel Adès, Jean-Luc Perfettini, Stéphane de Botton, Pierre Fenaux and Guido Kroemer
Pages 2139 - 2145
http://dx.doi.org/10.4161/cc.7.14.6268
Abstract | PDF

Malignant myeloblasts arising in high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are characterized by the constitutive activation of the anti-apoptotic transcription factor NF-κB. We found that DNA methyltransferase (DNMT) inhibitors (such as azacytidine and 5-aza-2'-deoxycytidine) and histone deacetylase (HDAC) inhibitors (such as trichostatin and valproic acid) efficiently induced apoptosis in the P39 MDS/AML cell line, correlating with an inhibition of NF-κB (which translocated from the nucleus to the cytoplasm). This effect was obtained rapidly, within a few hours, suggesting that it was not due to epigenetic reprogramming. Indeed, DNMT and HDAC inhibitors reduced the phosphorylation of the NF-κB-activating kinase IKKα/β, and this effect was also observed in enucleated cells. Finally, circulating myeloblasts from AML patients treated with the DNMT inhibitor 5-aza-2'-deoxycytidine manifested a rapid (2 hours post-treatment) inhibition of NF-κB and IKKα/β. Altogether, these results indicate that DNMT and HDAC inhibitors can inhibit the constitutive activation of NF-κB in malignant myeloblasts in vitro and in vivo through a novel mechanism.




REPORTS

Open Access Article

Effects of anti-microtubule agents on microtubule organization in cells lacking the kinesin-13 MCAK

David G. Hedrick, Jane R. Stout and Claire E. Walczak
Pages 2146 - 2156
http://dx.doi.org/10.4161/cc.7.14.6239
Abstract | PDF

Dynamic microtubules are necessary for proper mitotic spindle assembly and chromosome segregation during mitosis. Members of the kinesin superfamily of molecular motor proteins are important to spindle function. Of particular interest is the Kinesin-13 family member MCAK, which acts to regulate microtubule dynamics during spindle assembly and to ensure proper attachments of chromosomes to spindle microtubules. The unique ability of MCAK to regulate microtubule dynamics makes it a potential target for development of new drugs that alter spindle function. Here, we knocked down MCAK via RNAi in normal and malignant cell lines and found that the two tested malignant cell lines were acutely sensitive to MCAK knockdown, while the tested normal cells were less sensitive. In addition, we looked at the effect of combining MCAK knockdown and drug treatment with paclitaxel or vinblastine to identify spindle assembly defects. We found that MCAK knockdown increased the morphological defects of the microtubule cytoskeleton in HeLa cells caused by anti-microtubule drugs. Our studies support the idea that MCAK would be a good target for new chemotherapeutic development and may be particular useful in combination therapies with currently available anti-microtubule agents.

Open Access Article

Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1

Kerry-Ann Nakrieko, Alisa Vespa, David Mason, Timothy S. Irvine, Sudhir J. A. D'Souza and Lina Dagnino
Pages 2157 - 2166
http://dx.doi.org/10.4161/cc.7.14.6241
Abstract | PDF

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.

Open Access Article

Nurse cell of Trichinella spp. as a model of long-term cell cycle arrest

Magdalena Dabrowska, Marek Skoneczny, Zbigniew Zieliński and Wojciech Rode
Pages 2167 - 2178
http://dx.doi.org/10.4161/cc.7.14.6269
Abstract | PDF

Nurse cell (NC), formed from skeletal muscle cells upon infection with parasitic nematode trichina, presents a rare system of long-term suspension in the cell cycle. Signaling pathways and general biological functions of Trichinella spiralis NC, inferred from network analysis of competitive expression microarray data (NC vs. C2C12 myoblasts and myotubes), performed in Ingenuity Pathways Analysis (IPA) software and confirmed by Real-Time PCR, are presented. Assuming 4N DNA content in NC nuclei, its cell cycle arrest is identified herein as a hypermitogenic of G1-like type, accompanied by induction of senescence, underpinned by increased expression of p15, p16 and p57 cell cycle inhibitors, as well as overexpression of senescence-associated, β-galactosidase and numerous secretory factors. Growth factor signaling, with predominant role of EGF, cytokine signaling and G-protein-coupled receptor signaling, are suggested as dominant NC signal transduction pathways. Fos, FosB, STAT6, CREBL2, ID4 and retinoic acid dependent nuclear receptors appear to be the main factors determining NC specific gene transcription. Antigen presentation, complement signaling and β-amyloid processing pathways are also identified as operating in NC. In general, NC pathology is found to pertain to cancer, as well as other, including immunological and neurological, disorders.

Open Access Article

Cyclin A-CDK activity during G1 phase impairs MCM chromatin loading and inhibits DNA synthesis in mammalian cells

Leroy W. Wheeler, Nathan H. Lents and Joseph J. Baldassare
Pages 2179 - 2188
http://dx.doi.org/10.4161/cc.7.14.6270
Abstract | PDF

Progression through the mammalian cell division cycle is regulated by the sequential activation of cyclin-dependent kinases, CDKs, at specific phases of the cell cycle. Cyclin A-CDK2 and cyclin A-CDK1 phosphorylate nuclear substrates during S and G2 phases, respectfully. However, the DNA helicase complex, MCM2-7, is loaded onto the origin of replications in G1, prior to the normally scheduled induction of cyclin A. It has previously been shown that cyclin A-CDKs phosphorylate MCM2 and MCM4 in vitro, thereby diminishing helicase activity. Thus, in this study we hypothesize that, in vivo, cyclin A-CDK activity during G1 would result in an inhibition of progression into the S phase. To test this, we establish an in vivo method of inducing cyclin A-CDK activity in G1 phase and observe that activation of cyclin A-CDK, but not cyclin E-CDK complexes, inhibit DNA synthesis without affecting other G1 events such as cyclin D synthesis, E2F activation and cdc6 loading onto chromatin. We further report that the mechanism of this S phase inhibition occurs, at least in part, through impaired loading of MCM onto chromatin, presumably due to decreased levels of cdt1 and premature phosphorylation of MCM by cyclin A-CDK. In addition to providing in vivo confirmation of in vitro predictions regarding cyclin A-CDK phosphorylation of the MCM complex, our results provide insight into the cellular effects of unscheduled cyclin A-CDK activity in mammalian cells.

Open Access Article

Polypyrimidine tract binding protein regulates IRES-mediated translation of p53 isoforms

Richa Grover, Partho Sarothi Ray and Saumitra Das
Pages 2189 - 2198
http://dx.doi.org/10.4161/cc.7.14.6271
Abstract | PDF

The p53 tumor suppressor protein plays a key role in maintaining genomic integrity. Enhanced expression of p53 during genotoxic stress is due to both increased protein stability and translational up regulation. Previous reports have shown that p53 mRNA is translated from an alternative initiation codon to produce N-terminal truncated isoform (ΔN-p53) besides full-length p53. We have demonstrated that two internal ribosome entry sites (IRESs) regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. Here, we report that polypyrimidine tract-binding protein (PTB) is a p53 IRES interacting trans-acting factor. PTB protein binds specifically to both the p53 IRESs but with differential affinity. siRNA-mediated knockdown of PTB protein results in reduction of activity of both IRESs and also the levels of both the isoforms. It is well known that DNA-damaging agents such as doxorubicin enhance the expression of p53. Our results indicate that during doxorubicin treatment, PTB protein translocates from nucleus to the cytoplasm, probably to facilitate IRES mediated p53 translation. These observations suggest that the relative cytoplasmic abundance of PTB protein, under DNA-damaging conditions, might contribute to regulating the coordinated expression of the p53 isoforms, owing to the differential affinity of PTB binding to the two p53 IRESs.

Open Access Article

Aquaporin-4 expression is severely reduced in human sarcoglycanopathies and dysferlinopathies

Stefania Assereto, Mauro Mastrototaro, Silvia Stringara, Elisabetta Gazzerro, Paolo Broda, Grazia Paola Nicchia, Maria Svelto, Claudio Bruno, Vincenzo Nigro, M.P. Lisanti, Antonio Frigeri and Carlo Minetti
Pages 2199 - 2207
http://dx.doi.org/10.4161/cc.7.14.6272
Abstract | PDF

Aquaporin-4 (AQP4) is the major water channel expressed in fast-twitch skeletal muscle fibers. AQP4 is reduced in Duchenne and Becker Muscular Dystrophies, but not in caveolinopathies, thus suggesting an interaction with dystrophin or with members of the dystrophin-glycoprotein complex (DGC) rather than a nonspecific effect due to muscle membrane damage. To establish the role of sarcoglycans in AQP4 decrease occurring in muscular dystrophy, AQP4 expression was analyzed in muscle biopsies from patients affected by Limb Girdle Muscular Dystrophies (LGMDs) 2C-F genetically confirmed. In all the LGMD 2C-F (2α-, 1β-, 2γ-, 1δ-deficiency), AQP4 was severely decreased. This effect was associated to a marked reduction in α1-syntrophin levels. In control muscle AQP4 did not show a direct interaction with any of the four sarcoglycans but, it co-immunoprecipitated with α1-syntrophin, indicating that this modular protein may link AQP4 levels with the DGC complex. To determine whether AQP4 expression could be affected in other LGMDs due to the defect of a membrane protein not associated to the dystrophin complex, we examined AQP4 expression in 6 patients affected by dysferlin deficiency genetically confirmed. All the patients displayed a reduction of the water channel, and AQP4 expression appeared to correlate with the severity of the muscle histopathological lesions. However, differently from what observed in the sarcoglycans, α1-syntrophin expression was normal or just slightly reduced. These results seem to indicate an additional mechanism of regulation of AQP4 levels in muscle cells. In accordance with a specific effect of membrane muscle disorders, AQP4 protein levels were not changed in 3 mitochondrial and 3 metabolic myopathies. In conclusion, AQP4 expression and membrane localization are markedly reduced in LGMD 2B-2F. The role of AQP4 in the degenerative mechanism occurring in these diseases will be the object of our future research.

Open Access Article

Efficient adult skeletal muscle regeneration in mice deficient in p38β, p38γ and p38δ MAP kinases

Vanessa Ruiz-Bonilla, Eusebio Perdiguero, Lionel Gresh, Antonio L. Serrano, Mònica Zamora, Pedro Sousa-Victor, Mercè Jardí, Erwin F. Wagner and Pura Muñoz-Cánoves
Pages 2208 - 2214
http://dx.doi.org/10.4161/cc.7.14.6273
Abstract | PDF

Adult skeletal muscle is a very stable tissue containing a small population of myofiber-associated quiescent satellite cells compared with late embryonic/neonatal skeletal muscle, which contains highly proliferating myoblasts and small actively growing myofibers, suggesting that specific regulatory pathways may control myogenesis at distinct developmental stages. The p38 MAPK signaling pathway is central for myogenesis, based on studies using immortalized and neonatal primary myoblasts in vitro. However, the contribution of this pathway to adult myogenesis has never been investigated. Four p38 isoforms (p38α, p38β, p38γ and p38δ) exist in mammalian cells, being p38α and p38γ the most abundantly expressed isoforms in adult skeletal muscle. Given the embryonic/neonatal lethality of p38α-deficient mice, here we investigate the relative contribution of p38β, p38γ and p38δ to adult myogenesis. Regeneration and myofiber growth of adult muscle proceeds with similar efficiency in mice lacking p38β, p38γ and p38δ as in wild-type control mice. In agreement with this, there is no difference in adult satellite cell behavior in vitro among the different genotypes. Importantly, the pattern of p38 activation (ascribed to p38α) remains unperturbed during satellite myogenesis in vitro and adult muscle regeneration in wild type and p38β-, p38γ- and p38δ-deficient mice, rendering p38α as the essential p38 isoform sustaining adult myogenesis. This study constitutes the first analysis addressing the functionality of p38β, p38γ and p38δ in satellite cell-dependent adult muscle regeneration and growth.

Open Access Article

Distinct proliferative and transcriptional effects of the D-type cyclins in vivo

Lisa K. Mullany, Peter White, Eric A. Hanse, Christopher J. Nelsen, Melissa M. Goggin, Joseph E. Mullany, Chelsea K. Anttila, Linda E. Greenbaum, Klaus H. Kaestner and Jeffrey H. Albrecht
Pages 2215 - 2224
http://dx.doi.org/10.4161/cc.7.14.6274
Abstract | PDF

The D-type cyclins (D1, D2, and D3) are components of the cell cycle machinery and govern progression through G1 phase in response to extracellular signals. Although these proteins are highly homologous and conserved in evolution, they contain distinct structural motifs and are differentially regulated in various cell types. Cyclin D1 appears to play a role in many different types of cancer, whereas cyclins D2 and D3 are less frequently associated with malignancy. In this study, we transiently expressed cyclin D1, D2, or D3 in hepatocytes and analyzed transcriptional networks regulated by each. All three D-type cyclins promoted robust hepatocyte proliferation and marked liver growth, although cyclin D3 stimulated less DNA synthesis than D1 or D2. Accordingly, the three D-type cyclins similarly activated genes associated with cell division. Cyclin D1 regulated transcriptional pathways involved in the metabolism of carbohydrates, lipids, amino acids, and other substrates, whereas cyclin D2 did not regulate these pathways despite having an equivalent effect on proliferation. Comparison of transcriptional profiles following 70% partial hepatectomy and cyclin D1 transduction revealed a highly significant overlap, suggesting that cyclin D1 may regulate diverse cellular processes in the regenerating liver. In summary, these studies provide the first comparative analysis of the transcriptional networks regulated by the D-type cyclins and provide insight into novel functions of these key cell cycle proteins. Further study of the unique targets of cyclin D1 should provide further insight into its prominent role in proliferation, growth, and cancer.

Open Access Article

Differential regulation of centrosome integrity by DNA damage response proteins

Rekha Rai, Ashwini Phadnis, Sharda Haralkar, Rajender A. Badwe, Hui Dai, Kaiyi Li and Shiaw-Yih Lin
Pages 2225 - 2233
http://dx.doi.org/10.4161/cc.7.14.6303
Abstract | PDF

MDC1 and BRIT1 have been shown to function as key regulators in response to DNA damage. However, their roles in centrosomal regulation haven’t been elucidated. In this study, we demonstrated the novel functions of these two molecules in regulating centrosome duplication and mitosis. We found that MDC1 and BRIT1 were integral components of the centrosome that colocalize with γ-tubulin. Depletion of either protein led to centrosome amplification. However, the mechanisms that allow them to maintain centrosome integrity are different. MDC1-depleted cells exhibited centrosome overduplication, leading to multipolar mitosis, chromosome missegregation, and aneuploidy, whereas BRIT1 depletion led to misaligned spindles and/or lagging chromosomes with defective spindle checkpoint activation that resulted in defective cytokinesis and polyploidy. We further illustrated that both MDC1 and BRIT1 were negative regulators of Aurora A and Plk1, two centrosomal kinases involved in centrosome maturation and spindle assembly. Moreover, the levels of MDC1 and BRIT1 inversely correlated with centrosome amplification, defective mitosis, and cancer metastasis in human breast cancer. Together, MDC1 and BRIT1 may function as tumor-suppressor genes, at least in part by orchestrating proper centrosome duplication and mitotic spindle assembly.

Open Access Article

Moderate variations in CDC25B protein levels modulate the response to DNA damaging agents

Bernadette Aressy, Béatrix Bugler, Annie Valette, Denis Biard and Bernard Ducommun
Pages 2234 - 2240
http://dx.doi.org/10.4161/cc.7.14.6305
Abstract | PDF

CDC25B, one of the three members of the CDC25 dual-specificity phosphatase family, plays a critical role in the control of the cell cycle and in the checkpoint response to DNA damage. CDC25B is responsible for the initial dephosphorylation and activation of the cyclin-dependent kinases, thus initiating the train of events leading to entry into mitosis.1 The critical role played by CDC25B is illustrated by the fact that it is specifically required for checkpoint recovery2, 3 and that unscheduled accumulation of CDC25B is responsible for illegitimate entry into mitosis.3-5 Here, we report that in p53-/- colon carcinoma cells, a moderate increase in the CDC25B level is sufficient to impair the DNA damage checkpoint, to increase spontaneous mutagenesis, and to sensitize cells to ionising radiation and genotoxic agents. Using a tumour cell spheroid assay as an alternative to animal studies, we demonstrate that the level of CDC25B expression modulates growth inhibition and apoptotic death. Since CDC25B overexpression has been observed in a significant number of human cancers, including colon carcinoma, and is often associated with high grade tumours and poor prognosis1, our work suggests that the expression level of CDC25B might be a potential key parameter of the cellular response to cancer therapy.

Open Access Article

Signaling by dopamine regulates D2 receptors trafficking at the membrane

Emanuele Tirotta, Valerie Fontaine, Roberto Picetti, Monica Lombardi, Tarek A. Samad, Mustafa Oulad-Abdelghani, Robert Edwards and Emiliana Borrelli
Pages 2241 - 2248
http://dx.doi.org/10.4161/cc.7.14.6307
Abstract | PDF

sGi2 is a spliced variant of the GTP-binding protein Gαi2. By difference with Gαi2, which is mainly present at the plasma membrane, sGi2 is localized in intracellular compartments. The splicing event generates a novel C-terminal region in sGi2, which is necessary for its intracellular localization. The role of sGi2 is presently unknown, although its intracellular localization might underlie a possible role in the regulation of trafficking of 7TM receptors. Here, we show that sGi2 complexes with dopamine D2 receptors (D2R) in striatal neurons. The sGi2-D2R complex is readily observed in immunoprecipitation studies using specific antibodies for both proteins on mouse striatal extracts, which identify D2-specific bands of >80 KDa suggesting sGi2 interactions with D2R dimers. Importantly, the sGi2-D2R complex in the absence of receptor stimulation is mostly found in intracellular perinuclear areas in primary neuronal cultures. Treatment of neurons with quinpirole, a D2-specific agonist, results into diffusion of D2R and sGi2 staining throughout the cell and into neurites and membranes. This suggests that dopamine could regulate availability of D2 receptors at the cell surface. The formation of sGi2-D2R complex is mediated through the interaction of sGi2 with the third intracellular loop of D2Rs. As functional consequence of the D2R-sGi2 interaction, we observed a reduction of D2 binding sites at the plasma membrane, when the two proteins are co-expressed in transfected cells. Altogether these studies identify sGi2 as a D2R interacting protein involved in the regulation of D2R at the membrane through a dopamine mediated mechanism.

Open Access Article

Transposon-based mutagenesis identifies short RIP1 as an activator of NFκB

Maupali Dasgupta, Mukesh K. Agarwal, Patrick Varley, Tao Lu, George R. Stark and Eugene S. Kandel
Pages 2249 - 2256
http://dx.doi.org/10.4161/cc.7.14.6310
Abstract | PDF

We used a vector based on the Sleeping Beauty transposon to search for constitutive activators of NF-κB in cultured cells. Dominant mutations were produced by random insertion of a tetracycline-regulated promoter, which provided robust and exceptionally well-regulated expression of downstream genes. The ability to regulate the mutant phenotype was used to attribute the latter to the insertional event. In one such mutant, the promoter was inserted in the middle of the gene encoding receptor-interacting protein kinase 1 (RIP1). The protein encoded by the hybrid transcript lacks the putative kinase domain of RIP1, but potently stimulates NF-κB, AP-1 and Ets-1 activity. Similarly to TNFα treatment, expression of the short RIP1 was toxic to cells that failed to up-regulate NF-κB. The effects of short RIP1 did not require endogenous RIP1 or cytokine treatment and coincided with reduced responsiveness to TNFα. Additional evidence indicates that a similar short RIP1 could be produced naturally from the ripk1 locus. Interestingly, elevated expression of short RIP1 resulted in the loss of full length RIP1 from cells, pointing to a novel mechanism through which the abundance of RIP1 and the status of the related signaling cascades may be regulated.

Open Access Article

Caveolin-1 interacts with a lipid raft-associated population of fatty acid synthase

Dolores Di Vizio, Rosalyn M. Adam, Jayoung Kim, Robert Kim, Federica Sotgia, Terence Williams, Francesca Demichelis, Keith R. Solomon, Massimo Loda, Mark A. Rubin, Michael P. Lisanti and Michael R. Freeman
Pages 2257 - 2267
http://dx.doi.org/10.4161/cc.7.14.6475
Abstract | PDF

Fatty Acid Synthase (FASN), a cytoplasmic biosynthetic enzyme, is the major source of long-chain fatty acids, particularly palmitate. Caveolin-1 (Cav-1) is a palmitoylated lipid raft protein that plays a key role in signal transduction and cholesterol transport. Both proteins have been implicated in prostate cancer (PCa) progression, and Cav-1 regulates FASN expression in a mouse model of aggressive PCa. We demonstrate that FASN and Cav-1 are coordinately up-regulated in human prostate tumors in a hormone-insensitive manner. Levels of FASN and Cav-1 protein expression discriminated between localized and metastatic cancers, and the two proteins exhibited analogous subcellular locations in a tumor subset. Endogenous FASN and Cav-1 were reciprocally co-immunoprecipitated from human and murine PCa cells, indicating that FASN forms a complex with Cav-1. FASN, a cytoplasmic enzyme, was induced to associate transiently with lipid raft membranes following alterations in signal transduction within the Src, Akt and EGFR pathways, suggesting that co-localization of FASN and Cav-1 is dependent on activation of upstream signaling mediators. A Cav-1 palmitoylation mutant, Cav-1C133/143/156S, that prevents phosphorylation by Src, did not interact with FASN. When overexpressed in Cav-1-negative PCa cells, Cav-1C133/143/156S caused a reduction of both Src and Akt levels, as well as of their active, phosphorylated forms, in comparison with wild type Cav-1. These findings suggest that FASN and Cav-1 physically and functionally interact in PCa cells. They also imply that palmitoylation within this complex is involved in tumor growth and survival.




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Open Access Article

The XS domain of a plant specific SGS3 protein adopts a unique RNA recognition motif (RRM) fold

Dapeng Zhang and Vance L. Trudeau
Pages 2268 - 2270
http://dx.doi.org/10.4161/cc.7.14.6306
Abstract | PDF

No abstract available