Pleiotropy of the Drosophila JAK pathway cytokine Unpaired 3 in development and aging

The Janus kinase (JAK) pathway is an essential, highly re-utilized developmental signaling cascade found in most metazoans. In vertebrates, the JAK intracellular cascade mediates signaling by dozens of cytokines and growth factors. In Drosophila, the Unpaired (Upd) family, encoded by three tandemly duplicated genes, is the only class of ligands associated with JAK stimulation. Unpaired has a central role in activation of JAK for most pathway functions, while Unpaired 2 regulates body size through insulin signaling. We show here that the third member of the family, unpaired 3 (upd3), overlaps upd in expression in some tissues and is essential for a subset of JAK-mediated developmental functions. First, consistent with the known requirements of JAK signaling in gametogenesis, we find that mutants of upd3 show an age-dependent impairment of fertility in both sexes. In oogenesis, graded JAK activity stimulated by Upd specifies the fates of the somatic follicle cells. As upd3 mutant females age, defects arise that can be attributed to perturbations of the terminal follicle cells, which require the highest levels of JAK activation. Therefore, in oogenesis, the activities of Upd and Upd3 both appear to quantitatively contribute to specification of those follicle cell fates. Furthermore, the sensitization of upd3 mutants to age-related decline in fertility can be used to investigate reproductive senescence. Second, loss of Upd3 during imaginal development results in defects of adult structures, including reduced eye size and abnormal wing and haltere posture. The outstretched wing and small eye phenotypes resemble classical alleles referred to as outstretched (os) mutations that have been previously ascribed to upd. However, we show that os alleles affect expression of both upd and upd3 and map to untranscribed regions, suggesting that they disrupt regulatory elements shared by both genes. Thus the upd region serves as a genetically tractable model for coordinate regulation of tandemly duplicated gene families that are commonly found in higher eukaryotes.     

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.     

The Opposing Actions of Target of Rapamycin and AMP-Activated Protein Kinase in Cell Growth Control

Cell growth is a highly regulated, plastic process. Its control involves balancing positive regulation of anabolic processes with negative regulation of catabolic processes. Although target of rapamycin (TOR) is a major promoter of growth in response to nutrients and growth factors, AMP-activated protein kinase (AMPK) suppresses anabolic processes in response to energy stress. Both TOR and AMPK are conserved throughout eukaryotic evolution. Here, we review the fundamentally important roles of these two kinases in the regulation of cell growth with particular emphasis on their mutually antagonistic signaling.An efficient homeostatic response to maintain cellular energy despite a noncontinuous supply of nutrients is crucial for the survival of organisms. Cells have, therefore, evolved a host of molecular pathways to sense both intra- and extracellular nutrients and thereby quickly adapt their metabolism to changing conditions. The target of rapamycin (TOR) and AMP-activated protein kinase (AMPK) signaling pathways control growth and metabolism in a complementary manner with TOR promoting anabolic processes under nutrient- and energy-rich conditions, whereas AMPK promotes a catabolic response when cells are low on nutrients and energy. Both pathways are highly conserved from yeast to human. This review summarizes the cross talk between TOR and AMPK in different organisms.     

The regulation of INK4/ARF in cancer and aging

Loss of the INK4a/ARF/INK4b locus on chromosome 9p21 is among the most frequent cytogenetic events in human cancer. The products of the locus--p15(INK4b), p16(INK4a), and ARF--play widespread and independent roles in tumor suppression. Recent data also suggest that expression of p16(INK4a) induces an age-dependent decrease in the proliferative capacity of certain tissue-specific stem cells and unipotent progenitors. Here, we discuss the regulation and role of p16(INK4a), ARF, and p15(INK4b) in cancer and aging.     

雷帕霉素靶在小鼠受精卵早期发育中的作用研究

哺乳动物雷帕霉素靶(mTOR)是细胞生长的中心调控因子,应用RT-PCR、免疫印迹、放射性同位素体外测定酶活性等方法,研究mTOR在小鼠受精卵第一次有丝分裂过程中在卵中的表达、活性变化以及对卵裂的影响.研究发现mTOR在小鼠卵母细胞和受精卵中都有表达,在mRNA水平,mTOR从G2期开始降解,在蛋白水平,则各期没有明显变化;mTOR的激酶活性在受精后明显升高,并且在整个1-细胞期保持较高活性;mTOR的特异性抑制剂雷帕霉素能抑制卵裂,并且能抑制成熟促进因子MPF的调节亚基cyclin B的表达,从而抑制了MPF的活性.结果表明mTOR可能通过促进MPF的激活而促进小鼠受精卵的分裂.     

The Target of Rapamycin Signaling Pathway Regulates mRNA Turnover in the Yeast Saccharomyces cerevisiae

The target of rapamycin (TOR) signaling pathway is an important mechanism by which cell growth is regulated by nutrient availability in eukaryotes. We provide evidence that the TOR signaling pathway controls mRNA turnover in Saccharomyces cerevisiae. During nutrient limitation (diauxic shift) or after treatment with rapamycin (a specific inhibitor of TOR), multiple mRNAs were destabilized, whereas the decay of other mRNAs was unaffected. Our findings suggest that the regulation of mRNA decay by the TOR pathway may play a significant role in controlling gene expression in response to nutrient depletion. The inhibition of the TOR pathway accelerated the major mRNA decay mechanism in yeast, the deadenylation-dependent decapping pathway. Of the destabilized mRNAs, two different responses to rapamycin were observed. Some mRNAs were destabilized rapidly, while others were affected only after prolonged exposure. Our data suggest that the mRNAs that respond rapidly are destabilized because they have short poly(A) tails prematurely either as a result of rapid deadenylation or reduced polyadenylation. In contrast, the mRNAs that respond slowly are destabilized by rapid decapping. In summary, the control of mRNA turnover by the TOR pathway is complex in that it specifically regulates the decay of some mRNAs and not others and that it appears to control decay by multiple mechanisms.     

The coordinate regulation of pharyngeal development in C. elegans by lin-35/Rb, pha-1, and ubc-18

Organ development is a complex process involving the coordination of cell proliferation, differentiation, and morphogenetic events. Using a screen to identify genes that function coordinately with lin-35/Rb during animal development, we have isolated a weak loss-of-function (LOF) mutation in pha-1. lin-35; pha-1 double mutants are defective at an early step in pharyngeal morphogenesis leading to an abnormal pharyngeal architecture. pha-1 is also synthetically lethal with other class B synthetic multivulval (SynMuv) genes including the C. elegans E2F homolog, efl-1. Reporter analyses indicate that pha-1 is broadly expressed during embryonic development and that its functions reside in the cytoplasm. We also provide genetic and phenotypic evidence to support the model that PHA-1, a novel protein, and UBC-18, a ubiquitin-conjugating enzyme that we have previously shown to function with lin-35 during pharyngeal development, act in parallel pathways to regulate the activity of a common cellular target.     

Microtubule-associated Protein/Microtubule Affinity-regulating Kinase 4 (MARK4) Is a Negative Regulator of the Mammalian Target of Rapamycin Complex 1 (mTORC1)

The mammalian target of rapamycin (mTOR) is a central cell growth regulator. It resides in two protein complexes, which in mammals are referred to as mTORC1 and mTORC2. mTORC1, which is directly inhibited by rapamycin, promotes cell growth by stimulating protein synthesis and inhibiting autophagy. A wide range of extra and intracellular signals, including growth factors, nutrients, energy levels, and various stress conditions, regulates mTORC1. Dysregulation of mTORC1 contributes to many human diseases, including cancer, cardiovascular disease, autoimmunity, and metabolic disorder. In this study, we identified MARK4, an AMP-activated kinase-related kinase, as a negative regulator of mTORC1. In Drosophila S2 cells and mammalian cells, knockdown of MARK family member increased mTORC1 activity, whereas overexpression of MARK4 in mammalian cells significantly inhibited mTORC1 activity. Interestingly, MARK4 selectively inhibits mTORC1 activation by Rag GTPases, which are involved in amino acid signaling, but does not inhibit the effect of Rheb, which directly binds to and activates mTORC1. In addition, we found that MARK4 phosphorylates Raptor, a key component of mTORC1, and this phosphorylation may interfere with Raptor-Rag interaction. Our data demonstrate MARK4 as a new negative regulator of mTORC1.     

Inhibition of Target of Rapamycin Signaling and Stress Activate Autophagy in Chlamydomonas reinhardtii

Autophagy is a catabolic membrane-trafficking process whereby cells recycle cytosolic proteins and organelles under stress conditions or during development. This degradative process is mediated by autophagy-related (ATG) proteins that have been described in yeast, animals, and more recently in plants. In this study, we report the molecular characterization of autophagy in the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) is functionally conserved and may be used as a molecular autophagy marker. Like yeast ATG8, CrATG8 is cleaved at the carboxyl-terminal conserved glycine and is associated with membranes in Chlamydomonas. Cell aging or different stresses such as nutrient limitation, oxidative stress, or the accumulation of misfolded proteins in the endoplasmic reticulum caused an increase in CrATG8 abundance as well as the detection of modified forms of this protein, both landmarks of autophagy activation. Furthermore, rapamycin-mediated inhibition of the Target of Rapamycin signaling pathway, a major regulator of autophagy in eukaryotes, results in identical effects on CrATG8 and a relocalization of this protein in Chlamydomonas cells similar to the one observed upon nutrient limitation. Thus, our findings indicate that Chlamydomonas cells may respond to stress conditions by inducing autophagy via Target of Rapamycin signaling modulation.Protein turnover is essential for the adaptation of cells to variable environmental conditions. Similar to other eukaryotes, plants have developed two distinct mechanisms to regulate protein degradation, a selective ubiquitin/26S proteasome pathway (Vierstra, 2009) and macroautophagy (hereafter referred to as autophagy), a nonselective membrane-trafficking process (Bassham, 2009). During autophagy, a large number of cytosolic components, including entire organelles, organelle fragments, and protein complexes, are enclosed in bulk within a double-membrane structure known as the autophagosome and delivered to the vacuole/lysosome for degradation to recycle needed nutrients or degrade toxic components (Xie and Klionsky, 2007; Nakatogawa et al., 2009). The autophagosomes appear to arise from isolation membranes usually observed in close proximity to the vacuole called the preautophagosomal structure (PAS). These membranes expand and fuse to encircle portions of the cytoplasm, generating an autophagosome that is targeted to the vacuole. The outer membrane of the autophagosome then fuses with the vacuole membrane, and the remaining vesicle, known as the autophagic body, is finally released to the vacuole for its degradation (Xie and Klionsky, 2007).The evolutionary conservation of autophagy among eukaryotes indicates that structural and regulatory components of this cellular process must be also conserved. Accordingly, a significant number of autophagy-related (ATG) genes that participate in autophagy regulation and autophagosome formation have been identified, initially through genetic approaches in yeast and subsequently in higher eukaryotes, including mammals, insects, protozoa, and plants (Bassham et al., 2006; Bassham, 2007; Meijer et al., 2007). In yeast, two protein conjugation systems composed of the ubiquitin-like proteins ATG8 and ATG12 and the three enzymes ATG3, ATG7, and ATG10 play an essential role in autophagosome formation and seem to be conserved through evolution (Geng and Klionsky, 2008). ATG8 becomes modified with the lipid molecule phosphatidylethanolamine (PE) by the consecutive action of the ATG7 and ATG3 enzymes in a process mechanistically similar to ubiquitination (Ichimura et al., 2000). Prior to this modification, ATG8 must be cleaved by the Cys protease ATG4 to expose a C-terminal Gly residue that is conjugated to PE (Kirisako et al., 2000; Kim et al., 2001). ATG12 becomes covalently attached to the ATG5 protein in a conjugation reaction that is catalyzed by ATG7 and ATG10 (Mizushima et al., 1998). ATG8-PE and ATG12-ATG5 conjugates localize to autophagy-related membranes and are required for the initiation and expansion of autophagosomal membrane and hemifusion of this membrane with the vacuolar membrane (Hanada et al., 2007; Nakatogawa et al., 2007, 2009; Fujita et al., 2008; Geng and Klionsky, 2008; Xie et al., 2008).Our understanding of the autophagy process has substantially increased with the development of specific markers for autophagy. In plants, two markers for autophagosomes have been described, the monodansylcadaverine dye and GFP-ATG8 fusion protein (Yoshimoto et al., 2004; Contento et al., 2005; Thompson et al., 2005). As in other species, binding of ATG8 to autophagosomes has been used to monitor autophagy in plants. In contrast to yeast, where a single ATG8 gene is present, plants appear to contain a small gene family with several ATG8 isoforms, suggesting that autophagy is more complex in these photosynthetic organisms. For example, Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) encode nine and five ATG8 genes, respectively (Doelling et al., 2002; Hanaoka et al., 2002; Ketelaar et al., 2004; Chung et al., 2009). However, despite the high complexity of the ATG8-conjugating system in plants, important findings have been recently reported on the molecular characterization of autophagy using ATG8 as an autophagy marker in these organisms. The use of specific markers for autophagy in plants has revealed that this process is active at a basal level under normal growth and is induced upon nitrogen- or carbon-limiting conditions as well as in response to oxidative stress (Yoshimoto et al., 2004; Thompson et al., 2005; Xiong et al., 2005, 2007; Chung et al., 2009). Reverse genetic approaches have also been applied to a number of Arabidopsis ATG genes using T-DNA insertional mutants or RNA interference in order to investigate the physiological roles of autophagy in plants. The initial characterization of autophagy-deficient plants demonstrated that the ATG system is not essential under nutrient-rich conditions. However, a detailed analysis of these mutants indicated that autophagy is required for the proper response of the plant to nutrient limitation or pathogen infection. Plants lacking the ATG4, ATG5, ATG7, ATG9, or ATG10 gene display premature leaf senescence and are hypersensitive to nitrogen or carbon limitation (Doelling et al., 2002; Hanaoka et al., 2002; Yoshimoto et al., 2004; Thompson et al., 2005; Phillips et al., 2008). Arabidopsis plants with reduced levels of ATG18, which is required for autophagosome formation, are more sensitive to methyl viologen treatment and accumulate high levels of oxidized proteins, demonstrating that autophagic processes participate in the response of the plant to oxidative stress (Xiong et al., 2005, 2007). Plants deficient in the autophagy genes ATG6/Beclin1, ATG3, ATG7, and ATG9 exhibit unrestricted hypersensitive response lesions in response to pathogen infection (Liu et al., 2005; Hofius et al., 2009). These findings implicate autophagy as a prosurvival mechanism to restrict programmed cell death associated with the pathogen-induced hypersensitive response in plants. Arabidopsis ATG6 has also been shown to mediate pollen germination in a manner independent of autophagy (Fujiki et al., 2007).As mentioned above, autophagy is triggered among other factors by a reduction in the availability of nutrients. This starvation signal is transmitted to the autophagic machinery by important regulatory factors, including the Ser/Thr kinases Target of Rapamycin (TOR), ATG1, and SNF1 and the phosphatidylinositol 3-kinase ATG6/Beclin1 (Diaz-Troya et al., 2008b; Bassham, 2009; Cebollero and Reggiori, 2009). TOR has been identified as a negative regulator of autophagy in yeast, mammals, and fruit flies (Diaz-Troya et al., 2008b). The pharmacological inhibition of TOR by rapamycin leads to autophagy activation through a mechanism that requires the activation of the ATG1 kinase (Kamada et al., 2000). It has been recently demonstrated in mammals and fruit flies that a rapamycin-sensitive TOR signaling complex termed TORC1 directly phosphorylates and inhibits the ATG1 kinase and its regulatory protein ATG13 (Chang and Neufeld, 2009; Hosokawa et al., 2009; Jung et al., 2009). These regulatory proteins are conserved in plants, although except for ATG6 (Liu et al., 2005), there is no direct evidence for regulation of autophagy by these signaling pathways.The unicellular green alga Chlamydomonas reinhardtii has been used as a model for the study of important cellular and metabolic processes in photosynthetic organisms (Harris, 2001). More recently, Chlamydomonas has also been proposed as a useful system for the characterization of the TOR signaling pathway in photosynthetic eukaryotes based on the finding that, unlike plants, Chlamydomonas cell growth is sensitive to rapamycin (Crespo et al., 2005; Diaz-Troya et al., 2008a). Treatment of Chlamydomonas cells with rapamycin results in a pronounced increase of vacuole size that resembles autophagy-like processes (Crespo et al., 2005). However, a role of TOR in autophagy regulation could not be demonstrated due to the absence of an autophagy marker in Chlamydomonas. Actually, no studies have been reported on any autophagy-related protein in green algae, despite the high conservation of ATG genes in Chlamydomonas (Diaz-Troya et al., 2008b).This study reports the molecular and cellular characterization of autophagy in the green alga Chlamydomonas. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) may be used as a specific autophagy marker. Nutrient limitation and cell aging trigger an autophagic response that can be traced as an increase at the level of CrATG8, the detection of modified forms of CrATG8, and a change in the cellular localization of this protein. Furthermore, we demonstrate that autophagy is inhibited by a rapamycin-sensitive TOR cascade in Chlamydomonas.     

M(o)TOR of pseudo-hypoxic state in aging: Rapamycin to the rescue

A groundbreaking publication by Sinclair and coworkers has illuminated the pseudo-hypoxic state in aging and its reversibility. Remarkably, these data also fit the mTOR-centered model of aging. Here we discuss that the mTOR pathway can cause cellular pseudo-hypoxic state, manifested by HIF-1 expression and lactate production under normoxia. We found that rapamycin decreased HIF-1 and lactate levels in proliferating and senescent cells in vitro. This reduction was independent from mitochondrial respiration: rapamycin decreased lactate production in normoxia, hypoxia, and in the presence of the OXPHOS inhibitor oligomycin. We suggest that pseudo-hypoxic state is not necessarily caused by mitochondrial dysfunction, but instead mitochondrial dysfunction may be secondary to mTOR-driven hyperfunctions. Clinical applications of rapamycin for reversing pseudo-hypoxic state and lactate acidosis are discussed.