SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Modeling cAMP Oscilations - Self-organized and excitable signaling activities play important roles in a wide range of cellular functions in eukaryotic and prokaryotic cells. Cells require signaling networks to communicate amongst themselves, but also for response to environmental cues. Such signals involve complex spatial and temporal loops that may propagate as oscillations or waves. When Dictyostelium are starved for nutrients, cells within a territorial space secrete cAMP. Proximal cells move inward toward cAMP and relay the cAMP outward to recruit additional cells. To ensure directed inward movement, cells go through adapted and de-adapted states, for cAMP synthesis/degradation and directional cell movement, that oscillate at 6 min intervals. Developmental cAMP oscillations are characterized by a rise in cAMP synthesis and accumulation, followed by cessation of cAMP synthesis and increased cAMP degradation, with the cycle repeating with a defined temporal periodicity. Although many immediate components that regulate cAMP signaling (including receptors, G proteins, adenyl cyclase, phosphodiesterases, and kinases) are known, others are only inferred. Using biochemical experiments coupled with gene inactivation studies, we have identified new component members and model an integrated large (>25), multi-component kinetic pathway involving activation, inactivation (adaptation), re-activation (re-sensitization), feed-forward, and feed-back controls to generate developmental cAMP oscillations. Metabolomics - Changes in nutrients affect diverse cellular networks, making it challenging to distinguish metabolic paths that regulate growth from a switch to development. The life cycle of Dictyostelium is an excellent model to study metabolic signatures. Dictyostelium grow as single cells in nutrient-rich media, but, with nutrient withdrawal, growth ceases and cells enter multi-cell development. We developed conditions for rapid cell growth in rich-media, but where rapamycin-targeted inactivation of mTORC1 leads to a growth-to-development fate switch. We have shown that nutrient (glucose, amino acids) withdrawal significantly reduces many intermediates within most metabolic pathways, thus, negatively impacting glycolysis, the TCA cycle, pentose-phosphate shunt, etc. Rapamycin-induced development in the absence of nutrient withdrawal is expected to have a more limited influence on metabolic pathways. As part of the trans-NIH Metabolomics Consortium, we have undertaken time-course analyses of metabolomic changes in response to starvation- and rapamycin-induced development, to identify metabolic changes that are associated with a growth-to-development transition, but that are independent of nutrient depletion. We wish to identify metabolic changes that result form development (e.g. autophagic products), but also regulatory metabolites that may promote (e.g. AMP) or inhibit development. Initial results indicate that >5000 metabolite concentration differences are seen between starved-developed and growing cells, whereas <500 differ between growth and rapamycin-induced development in the absence of nutrient withdrawal. We anticipate identifying a defined catalog of metabolites whose concentrations are highly varied during development, independent of nutrient withdrawal. Some may function as epigenetic regulators of cell-fate change. As an example, a-ketoglutarate is a co-factor for dioxygenases that suppress repressive chromatin modifications with impact to transcription and a-ketoglutarate/succinate TCA component ratio differences can promote or suppress ES cell pluripotency. Our preliminary data in Dictyostelium indicate a >4-fold decrease in relative a ketoglutarate to succinate levels as development proceeds. We have further demonstrated that methyl-derivates of a ketoglutarate, for enhanced cell permeability, completely block developmental induction, even under starved conditions, without an effect on cell viability and growth. Data suggest that a ketoglutarate concentration is the primary effector, as developmental inhibition cannot be reversed with increased levels of succinate moieties. We will look for changes in the transcriptome and in chromatin organization that are specific to a-ketoglutarate treatment. Comparison of RNAseq data from cells starved in the presence or absence of exogenous a-ketoglutarate may further discriminate transcriptional changes closely associated with development from those only responsive to nutrient withdrawal. Growth-to-Development Developmental aggregation in Dictyostelium is lost at low cell density, but aggregation at non-permissive cell densities is rescued with secreted factor DPF1. Secreted DPF1 is synthesized as a larger precursor, single-pass transmembrane protein that is released by proteolytic cleavage and ectodomain shedding, leaving a 10 kDa transmembrane (TM) fragment. The TM/cytoplasmic domain of DPF1 possesses independent, cell autonomous activity for cell-substratum adhesion and cellular growth. We have created vectors that solely express the secreted or TM/cytoplasmic forms to understand the different functions. We have also identified a new gene DPF2, which is closely linked to DPF1, that encodes a sequence related protein with similar processing and ectodomain shedding properties. Ectodomain cleavage of both DPF1 and DPF2 is largely dependent upon calcium and calcium-dependent proteases (calpains). Secreted p150 kDa fragments of DPF1 and DPF2 have been purified in mg quantities and are being analyzed by MS/MS to map the specific cleavage sequences. We hypothesize there is pathway interaction between DPF1 and DPF2 and are testing this directly in mixing experiments with differentially epitope-tagged versions of DPF1 and DPF2. We have shown homo- and hetero-dimerization of the transmembrane domains of both DPF1 and DPF2. Lipid Storage during Fasting- Excessive cellular lipid storage can be a risk factor for metabolic disorders, including insulin resistance, cardiovascular disease, and hepatic steatosis. Intracellular lipid droplets are unique organelles that store metabolic precursors of cellular energy, membrane biosynthesis, steroid hormone synthesis, and signaling. The perilipins are a multi-protein family that targets lipid droplet surfaces and regulates lipid storage and hydrolysis. Plin2 binds hepatic LDs with expression levels that correlate with TAG content. We investigated the role of Plin2 in hepatic LD storage in fed and fasted plin2+/+ and plin2-/- mice. Chow-fed plin2-/- mice had comparable body weights, metabolic phenotype, glucose tolerance, and circulating TAG and total cholesterol levels to WT. Overnight fasting stimulates the degradation of stored adipose TAGs, with release of non-esterified fatty acids for circulation. Fasted plin2-/- mice showed substantially reduced accumulation of hepatic TAG compared to fasted WT. RNAseq revealed minor differences in hepatic gene expression between fed plin2+/+ and plin2-/- mice but marked differences in expression between fasted plin2+/+ and plin2-/- mice. Plin2 regulates hepatic lipid droplet size and accumulation of neutral lipid species in the fasted state. Hepatosteatosis - Recent studies describe transcriptome changes associated with hepatosteatosis, but it has been difficult to separate the effects on hepatic gene expression of fatty liver from that of obesity. We studied a plin2-/- mouse model, under conditions that are highly protective to hepatostaetosis, but not diet-induced obesity. We determined the mechanistic functions that protect plin2-/- livers from lipid accumulation and using RNAseq, compared hepatic transcriptomes of chow-fed or high-fat diet plin2+/+ and plin2 /- mice. We show that the Plin2 genotype, and accordingly hepatosteatosis, has a more limited impact on hepatic gene expression than does diet-induced obesity.