Apoptosis and Autophagy, Different Modes of Cell Death: How to Utilize Them to Fight Diseases?

A careful balance between cell death and survival is of key importance when it comes to the maintenance of cellular homeostasis [...].

Extracellular Vesicle-Mediated Metastasis Suppressors NME1 and NME2 Modify Lipid Metabolism in Fibroblasts.

Nowadays, extracellular vesicles (EVs) raise a great interest as they are implicated in intercellular communication between cancer and stromal cells. Our aim was to understand how vesicular NME1 and NME2 released by breast cancer cells influence the tumour microenvironment. As a model, we used human invasive breast carcinoma cells overexpressing NME1 or NME2, and first analysed in detail the presence of both isoforms in EV subtypes by capillary Western immunoassay (WES) and immunoelectron microscopy. Data obtained by both methods showed that NME1 was present in medium-sized EVs or microvesicles, whereas NME2 was abundant in both microvesicles and small-sized EVs or exosomes. Next, human skin-derived fibroblasts were treated with NME1 or NME2 containing EVs, and subsequently mRNA expression changes in fibroblasts were examined. RNAseq results showed that the expression of fatty acid and cholesterol metabolism-related genes was decreased significantly in response to NME1 or NME2 containing EV treatment. We found that FASN (fatty acid synthase) and ACSS2 (acyl-coenzyme A synthetase short-chain family member 2), related to fatty acid synthesis and oxidation, were underexpressed in NME1/2-EV-treated fibroblasts. Our data show an emerging link between NME-containing EVs and regulation of tumour metabolism.

Model systems in SDHx-related pheochromocytoma/paraganglioma.

Pheochromocytoma (PHEO) and paraganglioma (PGL) (together PPGL) are tumors with poor outcomes that arise from neuroendocrine cells in the adrenal gland, and sympathetic and parasympathetic ganglia outside the adrenal gland, respectively. Many follow germline mutations in genes coding for subunits of succinate dehydrogenase (SDH), a tetrameric enzyme in the tricarboxylic acid (TCA) cycle that both converts succinate to fumarate and participates in electron transport. Germline SDH subunit B (SDHB) mutations have a high metastatic potential. Herein, we review the spectrum of model organisms that have contributed hugely to our understanding of SDH dysfunction. In Saccharomyces cerevisiae (yeast), succinate accumulation inhibits alpha-ketoglutarate-dependent dioxygenase enzymes leading to DNA demethylation. In the worm Caenorhabditis elegans, mutated SDH creates developmental abnormalities, metabolic rewiring, an energy deficit and oxygen hypersensitivity (the latter is also found in Drosophila melanogaster). In the zebrafish Danio rerio, sdhb mutants display a shorter lifespan with defective energy metabolism. Recently, SDHB-deficient pheochromocytoma has been cultivated in xenografts and has generated cell lines, which can be traced back to a heterozygous SDHB-deficient rat. We propose that a combination of such models can be efficiently and effectively used in both pathophysiological studies and drug-screening projects in order to find novel strategies in PPGL treatment.

Sirtuins and Autophagy in Age-Associated Neurodegenerative Diseases: Lessons from the C. elegans Model.

Age-associated neurodegenerative diseases are known to have "impaired protein clearance" as one of the key features causing their onset and progression. Hence, homeostasis is the key to maintaining balance throughout the cellular system as an organism ages. Any imbalance in the protein clearance machinery is responsible for accumulation of unwanted proteins, leading to pathological consequences-manifesting in neurodegeneration and associated debilitating outcomes. Multiple processes are involved in regulating this phenomenon; however, failure to regulate the autophagic machinery is a critical process that hampers the protein clearing pathway, leading to neurodegeneration. Another important and widely known component that plays a role in modulating neurodegeneration is a class of proteins called sirtuins. These are class III histone deacetylases (HDACs) that are known to regulate various vital processes such as longevity, genomic stability, transcription and DNA repair. These enzymes are also known to modulate neurodegeneration in an autophagy-dependent manner. Considering its genetic relevance and ease of studying disease-related endpoints in neurodegeneration, the model system Caenorhabditis elegans has been successfully employed in deciphering various functional outcomes related to critical protein molecules, cell death pathways and their association with ageing. This review summarizes the vital role of sirtuins and autophagy in ageing and neurodegeneration, in particular highlighting the knowledge obtained using the C. elegans model system.

Conserved and Distinct Elements of Phagocytosis in Human and C. elegans.

Endocytosis provides the cellular nutrition and homeostasis of organisms, but pathogens often take advantage of this entry point to infect host cells. This is counteracted by phagocytosis that plays a key role in the protection against invading microbes both during the initial engulfment of pathogens and in the clearance of infected cells. Phagocytic cells balance two vital functions: preventing the accumulation of cell corpses to avoid pathological inflammation and autoimmunity, whilst maintaining host defence. In this review, we compare elements of phagocytosis in mammals and the nematode Caenorhabditis elegans. Initial recognition of infection requires different mechanisms. In mammals, pattern recognition receptors bind pathogens directly, whereas activation of the innate immune response in the nematode rather relies on the detection of cellular damage. In contrast, molecules involved in efferocytosis-the engulfment and elimination of dying cells and cell debris-are highly conserved between the two species. Therefore, C. elegans is a powerful model to research mechanisms of the phagocytic machinery. Finally, we show that both mammalian and worm studies help to understand how the two phagocytic functions are interconnected: emerging data suggest the activation of innate immunity as a consequence of defective apoptotic cell clearance.

The SDHB Arg230His mutation causing familial paraganglioma alters glycolysis in a new Caenorhabditis elegans model.

The conserved B-subunit of succinate dehydrogenase (SDH) participates in the tricarboxylic acid cycle (TCA) cycle and mitochondrial electron transport. The Arg230His mutation in SDHB causes heritable pheochromocytoma/paraganglioma (PPGL). In Caenorhabditiselegans, we generated an in vivo PPGL model (SDHB-1 Arg244His; equivalent to human Arg230His), which manifests delayed development, shortened lifespan, attenuated ATP production and reduced mitochondrial number. Although succinate is elevated in both missense and null sdhb-1(gk165) mutants, transcriptomic comparison suggests very different causal mechanisms that are supported by metabolic analysis, whereby only Arg244His (not null) worms demonstrate elevated lactate/pyruvate levels, pointing to a missense-induced, Warburg-like aberrant glycolysis. In silico predictions of the SDHA-B dimer structure demonstrate that Arg230His modifies the catalytic cleft despite the latter's remoteness from the mutation site. We hypothesize that the Arg230His SDHB mutation rewires metabolism, reminiscent of metabolic reprogramming in cancer. Our tractable model provides a novel tool to investigate the metastatic propensity of this familial cancer and our approach could illuminate wider SDH pathology.This article has an associated First Person interview with the first author of the paper.

The Function of NM23-H1/NME1 and Its Homologs in Major Processes Linked to Metastasis.

Metastasis suppressor genes (MSGs) inhibit different biological processes during metastatic progression without globally influencing development of the primary tumor. The first MSG, NM23 (non-metastatic clone 23, isoform H1) or now called NME1 (stands for non-metastatic) was identified some decades ago. Since then, ten human NM23 paralogs forming two groups have been discovered. Group I NM23 genes encode enzymes with evolutionarily highly conserved nucleoside diphosphate kinase (NDPK) activity. In this review we summarize how results from NDPKs in model organisms converged on human NM23 studies. Next, we examine the role of NM23-H1 and its homologs within the metastatic cascade, e.g. cell migration and invasion, proliferation and apoptosis. NM23-H1 homologs are well known inhibitors of cell migration. Drosophila studies revealed that AWD, the fly counterpart of NM23-H1 is a negative regulator of cell motility by modulating endocytosis of chemotactic receptors on the surface of migrating cells in cooperation with Shibire/Dynamin; this mechanism has been recently confirmed by human studies. NM23-H1 inhibits proliferation of tumor cells by phosphorylating the MAPK scaffold, kinase suppressor of Ras (KSR), resulting in suppression of MAPK signalling. This mechanism was also observed with the C. elegans homolog, NDK-1, albeit with an inverse effect on MAPK activation. Both NM23-H1 and NDK-1 promote apoptotic cell death. In addition, NDK-1, NM23-H1 and their mouse counterpart NM23-M1 were shown to promote phagocytosis in an evolutionarily conserved manner. In summary, inhibition of cell migration and proliferation, alongside actions in apoptosis and phagocytosis are all mechanisms through which NM23-H1 acts against metastatic progression.

The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/Dynamin.

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.

Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells.

BACKGROUND: Glioma is the most common highly aggressive, primary adult brain tumour. Clinical data show that therapeutic approaches cannot reach the expectations in patients, thus gliomas are mainly incurable diseases. Tumour cells can adapt rapidly to alterations during therapeutic treatments related to their metabolic rewiring and profound heterogeneity in tissue environment. Renewed interests aim to develop effective treatments targeting angiogenesis, kinase activity and/or cellular metabolism. mTOR (mammalian target of rapamycin), whose hyper-activation is characteristic for many tumours, promotes metabolic alterations, macromolecule biosynthesis, cellular growth and survival. Unfortunately, mTOR inhibitors with their lower toxicity have not resulted in appreciable survival benefit. Analysing mTOR inhibitor sensitivity, other metabolism targeting treatments and their combinations could help to find potential agents and biomarkers for therapeutic development in glioma patients. METHODS: In vitro proliferation assays, protein expression and metabolite concentration analyses were used to study the effects of mTOR inhibitors, other metabolic treatments and their combinations in glioma cell lines. Furthermore, mTOR activity and cellular metabolism related protein expression patterns were also investigated by immunohistochemistry in human biopsies. Temozolomide and/or rapamycin treatments altered the expressions of enzymes related to lipid synthesis, glycolysis and mitochondrial functions as consequences of metabolic adaptation; therefore, other anti-metabolic drugs (chloroquine, etomoxir, doxycycline) were combined in vitro. RESULTS: Our results suggest that co-targeting metabolic pathways had tumour cell dependent additive/synergistic effects related to mTOR and metabolic protein expression patterns cell line dependently. Drug combinations, especially rapamycin + doxycycline may have promising anti-tumour effect in gliomas. Additionally, our immunohistochemistry results suggest that metabolic and mTOR activity alterations are not related to the recent glioma classification, and these protein expression profiles show individual differences in patients' materials. CONCLUSIONS: Based on these, combinations of different new/old drugs targeting cellular metabolism could be promising to inhibit high adaptation capacity of tumour cells depending on their metabolic shifts. Relating to this, such a development of current therapy needs to find special biomarkers to characterise metabolic heterogeneity of gliomas.

Developmentally regulated autophagy is required for eye formation in Drosophila.

The compound eye of the fruit fly Drosophila melanogaster is one of the most intensively studied and best understood model organs in the field of developmental genetics. Herein we demonstrate that autophagy, an evolutionarily conserved selfdegradation process of eukaryotic cells, is essential for eye development in this organism. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (Atg) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. We also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog Lab (Labial), a master regulator of early development. Atg8a transcription is repressed in front of, while activated along, the MF by Lab. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought. ABBREVIATIONS: αTub84B, α-Tubulin at 84B; Act5C, Actin5C; AO, acridine orange; Atg, autophagy-related; Ato, Atonal; CASP3, caspase 3; Dcr-2; Dicer-2; Dfd, Deformed; DZ, differentiation zone; eGFP, enhanced green fluorescent protein; EM, electron microscopy; exd, extradenticle; ey, eyeless; FLP, flippase recombinase; FRT, FLP recognition target; Gal4, gene encoding the yeast transcription activator protein GAL4; GFP, green fluorescent protein; GMR, Glass multimer reporter; Hox, homeobox; hth, homothorax; lab, labial; L3F, L3 feeding larval stage; L3W, L3 wandering larval stage; lf, loss-of-function; MAP1LC3, microtubule-associated protein 1 light chain 3; MF, morphogenetic furrow; PE, phosphatidylethanolamine; PBS, phosphate-buffered saline; PI3K/PtdIns3K, class III phosphatidylinositol 3-kinase; PZ, proliferation zone; Ref(2)P, refractory to sigma P, RFP, red fluorescent protein; RNAi, RNA interference; RpL32, Ribosomal protein L32; RT-PCR, reverse transcription-coupled polymerase chain reaction; S.D., standard deviation; SQSTM1, Sequestosome-1, Tor, Target of rapamycin; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay; UAS, upstream activation sequence; qPCR, quantitative real-time polymerase chain reaction; w, white.

Sex-specific regulation of aging in Caenorhabditis elegans.

A fascinating aspect of sexual dimorphism in various animal species is that the two sexes differ substantially in lifespan. In humans, for example, women's life expectancy exceeds that of men by 3-7 years. Whether this trait can be attributed to dissimilar lifestyles or genetic (regulatory) factors remains to be elucidated. Herein, we demonstrate that in the nematode Caenorhabditis elegans, the significantly longer lifespan of hermaphrodites-which are essentially females capable of sperm production-over males is established by TRA-1, the terminal effector of the sex-determination pathway. This transcription factor directly controls the expression of daf-16/FOXO, which functions as a major target of insulin/IGF-1 signaling (IIS) and key modulator of aging across diverse animal phyla. TRA-1 extends hermaphrodite lifespan through promoting daf-16 activity. Furthermore, TRA-1 also influences reproductive growth in a DAF-16-dependent manner. Thus, the sex-determination machinery is an important regulator of IIS in this organism. These findings provide a mechanistic insight into how longevity and development are specified unequally in the two genders. As TRA-1 is orthologous to mammalian GLI (glioma-associated) proteins, a similar sex-specific mechanism may also operate in humans to determine lifespan.

The dosage-dependent effect exerted by the NM23-H1/H2 homolog NDK-1 on distal tip cell migration in C. elegans.

Abnormal regulation of cell migration and altered rearrangement of the cytoskeleton are fundamental properties of metastatic cells. The first identified metastasis suppressor NM23-H1, which displays nucleoside-diphosphate kinase (NDPK) activity is involved in these processes. NM23-H1 inhibits the migratory and invasive potential of some cancer cells. Correspondingly, numerous invasive cancer cell lines (eg, breast, colon, oral, hepatocellular carcinoma, and melanoma) display low endogenous NM23 levels. In this review, we summarize mechanisms, which are linked to the anti-metastatic activity of NM23. In human cancer cell lines NM23-H1 was shown to regulate cytoskeleton dynamics through inactivation of Rho/Rac-type GTPases. The Drosophila melanogaster NM23 homolog abnormal wing disc (AWD) controls tracheal and border cell migration. The molecular function of AWD is well characterized in both processes as a GTP supplier of Shi/Dynamin whereby AWD regulates the level of chemotactic receptors on the surface of migrating cells through receptor internalization, by its endocytic function. Our group studied the role of the sole group I NDPK, NDK-1 in distal tip cell (DTC) migration in Caenorhabditis elegans. In the absence of NDK-1 the migration of DTCs is incomplete. A half dosage of NDPK as present in ndk-1 (+/-) heterozygotes results in extra turns and overshoots of migrating gonad arms. Conversely, an elevated NDPK level also leads to incomplete gonadal migration owing to a premature stop of DTCs in the third phase of migration, where NDK-1 acts. We propose that NDK-1 exerts a dosage-dependent effect on the migration of DTCs. Our data derived from DTC migration in C. elegans is consistent with data on AWD's function in Drosophila. The combined data suggest that NDPK enzymes control the availability of surface receptors to regulate cell-sensing cues during cell migration. The dosage of NDPKs may be a coupling factor in cell migration by modulating the efficiency of receptor recycling.

The relationship between reproductive and biochemical ageing at the time of the menopausal transition.

The biochemical ageing status of women in the menopausal transition was studied using quantitative analysis of age- and autophagy-related gene activities (CDC42 and MAP1LC3 genes were selected as target genes). Free estradiol and progesterone levels in saliva were estimated. General linear models were used to determine the relationship between lifestyle, health status, socioeconomic factors and CDC42 and MAP1LC3 gene expression levels. Gene expression analysis revealed (1) an increasing expression of CDC42 gene after 45years in women, (2) expression level of CDC42 gene associated with menopausal status, (3) while endocrine status was found to associate with the expression of both of the studied age-related genes, (4) the "never used hormonal contraceptives" and "obese nutritional status" were the strongest factors for increased level of age-related gene expressions, and (5) changes in gene expression levels by ageing should be studied by considering not only chronological, but also biological ages. Gene expression profile of ageing has mostly been studied in model systems or human blood samples, but rarely in human saliva samples. The concordance of results between the present and former gene expression analyses, and the simplicity of saliva sample collection emphasizes the importance of saliva tissue samples in gene expression analyses especially in epidemiological surveys.

Nucleoside diphosphate kinases (NDPKs) in animal development.

In textbooks of biochemistry, nucleoside diphosphate conversion to a triphosphate by nucleoside diphosphate 'kinases' (NDPKs, also named NME or NM23 proteins) merits a few lines of text. Yet this essential metabolic function, mediated by a multimeric phosphotransferase protein, has effects that lie beyond a simple housekeeping role. NDPKs attracted more attention when NM23-H1 was identified as the first metastasis suppressor gene. In this review, we examine these NDPK enzymes from a developmental perspective because of the tractable phenotypes found in simple animal models that point to common themes. The data suggest that NDPK enzymes control the availability of surface receptors to regulate cell-sensing cues during cell migration. NDPKs regulate different forms of membrane enclosure that engulf dying cells during development. We suggest that NDPK enzymes have been essential for the regulated uptake of objects such as bacteria or micronutrients, and this evolutionarily conserved endocytic function contributes to their activity towards the regulation of metastasis.

The metastasis suppressor Nm23 as a modulator of Ras/ERK signaling.

NM23-H1 (also known as NME1) was the first identified metastasis suppressor, which displays a nucleoside diphosphate kinase (NDPK) and histidine protein kinase activity. NDPKs are linked to many processes, such as cell migration, proliferation, differentiation, but the exact mechanism whereby NM23-H1 inhibits the metastatic potential of cancer cells remains elusive. However, some recent data suggest that NM23-H1 may exert its anti-metastatic effect by blocking Ras/ERK signaling. In mammalian cell lines NDPK-mediated attenuation of Ras/ERK signaling occurs through phosphorylation (thus inactivation) of KSR (kinase suppressor of Ras) scaffolds. In this review I summarize our knowledge about KSR's function and its regulation in mammals and in C. elegans. Genetic studies in the nematode contributed substantially to our understanding of the function and regulation of the Ras pathway (i.e. KSR's discovery is also linked to the nematode). Components of the RTK/Ras/ERK pathway seem to be highly conserved between mammals and worms. NDK-1, the worm homolog of NM23-H1 affects Ras/MAPK signaling at the level of KSRs, and a functional interaction between NDK-1/NDPK and KSRs was first demonstrated in the worm in vivo. However, NDK-1 is a factor, which is necessary for proper MAPK activation, thus it activates rather than suppresses Ras/MAPK signaling in the worm. The contradiction between results in mammalian cell lines and in the worm regarding NDPKs' effect exerted on the outcome of Ras signaling might be resolved, if we better understand the function, structure and regulation of KSR scaffolds.

NDK-1, the homolog of NM23-H1/H2 regulates cell migration and apoptotic engulfment in C. elegans.

Abnormal regulation of cell migration and altered rearrangement of cytoskeleton are characteristic of metastatic cells. The first described suppressor of metastatic processes is NM23-H1, which displays NDPK (nucleoside-diphosphate kinase) activity. To better understand the role of nm23 genes in cell migration, we investigated the function of NDK-1, the sole Caenorhabditis elegans homolog of group I NDPKs in distal tip cell (DTC) migration. Dorsal phase of DTC migration is regulated by integrin mediated signaling. We find that ndk-1 loss of function mutants show defects in this phase. Epistasis analysis using mutants of the α-integrin ina-1 and the downstream functioning motility-promoting signaling module (referred to as CED-10 pathway) placed NDK-1 downstream of CED-10/Rac. As DTC migration and engulfment of apoptotic corpses are analogous processes, both partially regulated by the CED-10 pathway, we investigated defects of apoptosis in ndk-1 mutants. Embryos and germ cells defective for NDK-1 showed an accumulation of apoptotic cell corpses. Furthermore, NDK-1::GFP is expressed in gonadal sheath cells, specialized cells for engulfment and clearence of apoptotic corpses in germ line, which indicates a role for NDK-1 in apoptotic corpse removal. In addition to the CED-10 pathway, engulfment in the worm is also mediated by the CED-1 pathway. abl-1/Abl and abi-1/Abi, which function in parallel to both CED-10/CED-1 pathways, also regulate engulfment and DTC migration. ndk-1(-);abi-1(-) double mutant embryos display an additive phenotype (e. g. enhanced number of apoptotic corpses) which suggests that ndk-1 acts in parallel to abi-1. Corpse number in ndk-1(-);ced-10(-) double mutants, however, is similar to ced-10(-) single mutants, suggesting that ndk-1 acts downstream of ced-10 during engulfment. In addition, NDK-1 shows a genetic interaction with DYN-1/dynamin, a downstream component of the CED-1 pathway. In summary, we propose that NDK-1/NDPK might represent a converging point of CED-10 and CED-1 pathways in the process of cytoskeleton rearrangement.

The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans.

The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis.

Identification of novel cis-regulatory regions from the Notch receptor genes lin-12 and glp-1 of Caenorhabditis elegans.

Notch signaling regulates various cellular processes such as growth, proliferation and differentiation, and plays a key role in tissue patterning during animal development. In humans, defects in Notch signaling have been implicated in cancer, stroke, neurodegeneration, as well as learning and memory deficits. The genome of the nematode Caenorhabditis elegans encodes two members of the Notch transmembrane receptor family, LIN-12 and GLP-1, which have both unique and shared developmental functions. LIN-12 affects diverse cell fate specification events at certain embryonic and larval stages, including the ABplp lineage (a neuronal precursor), intestinal primordium, gonadal anchor cell and secondary vulval precursor cells. In addition to developmental functions, it also operates in the adult nervous system to control locomotion, memory and chemosensory response. Although lin-12 expression was subjected to intense analysis, it was almost not demonstrable in neurons; occasional lin-12 expression was detected only in the two RIG interneurons of young larvae. Here we identify two cis-regulatory regions from lin-12, both of them are specified by the presence of a conserved EXD/HOX composite binding site. One of these regions is located in the first intron and required for driving transgene expression in vulval precursor cell lineages and specific gonadal cells. The other region is located in the second intron and can confer neuronal expression for lin-12 throughout life. The latter regulatory element is highly conserved in the paralogous glp-1 genomic environment, suggesting redundant developmental and physiological roles for the two Notch paralogs in the C. elegans nervous system.

Heat shock factor-1 intertwines insulin/IGF-1, TGF-ß and cGMP signaling to control development and aging.

BACKGROUND: Temperature affects virtually all cellular processes. A quick increase in temperature challenges the cells to undergo a heat shock response to maintain cellular homeostasis. Heat shock factor-1 (HSF-1) functions as a major player in this response as it activates the transcription of genes coding for molecular chaperones (also called heat shock proteins) that maintain structural integrity of proteins. However, the mechanisms by which HSF-1 adjusts fundamental cellular processes such as growth, proliferation, differentiation and aging to the ambient temperature remain largely unknown. RESULTS: We demonstrate here that in Caenorhabditis elegans HSF-1 represses the expression of daf-7 encoding a TGF-ß (transforming growth factor-beta) ligand, to induce young larvae to enter the dauer stage, a developmentally arrested, non-feeding, highly stress-resistant, long-lived larval form triggered by crowding and starvation. Under favorable conditions, HSF-1 is inhibited by crowding pheromone-sensitive guanylate cyclase/cGMP (cyclic guanosine monophosphate) and systemic nutrient-sensing insulin/IGF-1 (insulin-like growth factor-1) signaling; loss of HSF-1 activity allows DAF-7 to promote reproductive growth. Thus, HSF-1 interconnects the insulin/IGF-1, TGF-ß and cGMP neuroendocrine systems to control development and longevity in response to diverse environmental stimuli. Furthermore, HSF-1 upregulates another TGF-ß pathway-interacting gene, daf-9/cytochrome P450, thereby fine-tuning the decision between normal growth and dauer formation. CONCLUSION: Together, these results provide mechanistic insight into how temperature, nutrient availability and population density coordinately influence development, lifespan, behavior and stress response through HSF-1.

Shared developmental roles and transcriptional control of autophagy and apoptosis in Caenorhabditis elegans.

Autophagy is a lysosome-mediated self-degradation process of eukaryotic cells that, depending on the cellular milieu, can either promote survival or act as an alternative mechanism of programmed cell death (PCD) in terminally differentiated cells. Despite the important developmental and medical implications of autophagy and the main form of PCD, apoptosis, orchestration of their regulation remains poorly understood. Here, we show in the nematode Caenorhabditis elegans, that various genetic and pharmacological interventions causing embryonic lethality trigger a massive cell death response that has both autophagic and apoptotic features. The two degradation processes are also redundantly required for normal development and viability in this organism. Furthermore, the CES-2-like basic region leucine-zipper (bZip) transcription factor ATF-2, an upstream modulator of the core apoptotic cell death pathway, is able to directly regulate the expression of at least two key autophagy-related genes, bec-1/ATG6 and lgg-1/ATG8. Thus, the two cell death mechanisms share a common method of transcriptional regulation. Together, these results imply that under certain physiological and pathological conditions, autophagy and apoptosis are co-regulated to ensure the proper morphogenesis and survival of the developing organism. The identification of apoptosis and autophagy as compensatory cellular pathways in C. elegans might help us to understand how dysregulated PCD in humans can lead to diverse pathologies, including cancer, neurodegeneration and diabetes.