SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Metabolomics - Changes in nutrient levels affect diverse cellular networks, making it challenging to distinguish metabolic paths that regulate growth or a switch to development. The life cycle of Dictyostelium presents an excellent model to such study metabolic signatures. Dictyostelium grow as single cells in nutrient-rich media, but, with nutrient withdrawal, growth ceases and cells enter multi-cell development. Kinases mTORC1 and AMPK are reciprocal energy sensors, to control cellular growth; mTORC1 is required for cell growth, activated by nutrients, and inactivated upon nutrient withdrawal. AMPK has opposite kinetics. In addition, mTORC1 and AMPK are negative regulators of each other, reinforcing reciprocal patterns. While both respond to specific metabolites (e.g. amino acids, AMP) they also control metabolic pathways. We have shown that rapamycin-targeted inactivation of mTORC1, in the absence of nutrient withdrawal, leads to reciprocal activation of AMPK, which is sufficient to effect a growth-to-developmental fate switch and to induce multi-cell development of Dictyostelium. Using an RNA-sequencing approach, we then identified mTORC1/AMPK-regulated transcriptional networks and associated signaling pathways that are essential for early developmental induction, but that are regulated independently of nutrient withdrawal. We use this system as part of The trans-NIH Metabolomics Consortium to identify metabolic changes that are associated with a growth-to-development transition, but that are not dependent of nutrient depletion. Preliminary data indicate a >4-fold increase in relative succinate to alpha ketoglutarate levels, as cells enter development and treatment with exogenous alpha ketoglutarate blocks development; molecular targets are being investigated. We also collaborate with Dr. Vivek Garg at the University of Maryland School of Medicine on related mitochondrial Ca+2 regulatory modes. Growth-to-Development Fate Decisions - 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 both cAMP synthesis/degradation and directional cell movement, that oscillate at 6 min intervals. Such cell-cell interaction responses are enhanced by increased cell density. We had identified a secreted 150 kDa protein, termed DPF (Development Promoting Factor), which possesses density dependent activity. Cells that overexpress DPF develop at low cell densities; cells deficient for DPF require higher cell density, compared to WT. DPF is synthesized as a larger precursor, single-pass transmembrane protein that is released by proteolytic cleavage and ectodomain shedding. The secreted 150 kDa factor is sufficient to regulate development non-autonomously, but the TM/cytoplasmic domain of DPF possesses independent cell autonomous activity for cell-substratum adhesion and cellular growth. We have created vectors that express only the secreted or TM/cytoplasmic forms to test mechanisms. We have also identified a sequence related protein with similar DPF properties and potential pathway. The phenotypes of individual-null cells are not identical but are consistent with a model where interactions mediate cell density sensing and development. Such a model is being tested directly in cell mixing experiments with WT and mutant proteins. We have also shown that DPF ectodomain cleavage is largely inhibited by depletion of calcium and by mutation of calcium-dependent proteases (calpains). The cleaved factor has been purified and is being analyzed by MS/MS to identify the cleavage sequence. To understand the non-autonomous effects of DPF on heterologous cell gene expression, we have prepared single-cell RNAseq time courses, comparing 100% DPF-nulls to 99% null/1% over-expressor mixes. SC RNAseq we enable us to unequivocally identify cells that do not express endogenous DPF and to determine how they specifically respond to exogenously added factor. Regulation of mRNA Stability - Post-transcriptional processes mediated by mRNA binding proteins represent important control points in gene expression. We have identified a tandem zinc finger protein TTP in Dictyostelium that promotes mRNA decay, likely through interaction with CCR4-NOT protein complexes. Six mRNA transcripts are consistently upregulated in null and point mutants compared to WT, and the 3'-untranslated regions (3'-UTRs) of all six contain multiple, overlapping UUAUUUAUU motifs; one 3'-UTR conferred TTP post-transcriptional stability regulation to a heterologous mRNA that was abrogated by mRNA mutations in the core UA-rich motifs. Expression of the Dictyostelium protein in mammalian cells promotes mRNA decay of mammalian transcripts that contain these motifs, despite evolutionary divergence of nearly a billion years. Function requires more than a single motif. Dependent may also require open structures within the 3UTRs. Interactions with mRNAs and proteins are being followed with IP of functionally active epitope tagged TTP. Regulation of Lipid Storage - 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. Perlipin 5 (Plin5) is abundantly expressed in the heart where it binds to lipid droplets (LDs) and facilitates physical interaction between LDs and mitochondria. We studied fatty acid uptake, LD accumulation, fatty acid oxidation, and tolerance to hypoxia in isolated cardiomyocytes from adult Plin5+/+ and Plin5-/-. Plin5-/- cardiomyocytes stored less LDs than did Plin5+/+, which was reversed by inhibition of the adipocyte triglyceride lipase (ATGL). Pre-incubation of Plin5-/- cardiomyocytes with oleic acids, increased glycogen and improved tolerance to hypoxia, by normalizing ventricular pressure and infarct size following ischemia. Plin5 seems to facilitate cardiac LD storage primarily by repressing adipose triglyceride lipase activity, without altering cardiac fatty acid oxidation capacity. Expression of Plin5 and cardiac LD content in isolated cardiomyocytes has little importance for tolerance to acute hypoxia and ischemia, which contrasts the protective role for Plin5 in actual mouse models during myocardial ischemia, emphasizing their more complex metabolic. Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. Steroidogenic cells primarily store CEs in LDs, as contrasted to the majority of mammalian cell types that predominantly store triacylglycerol (TAG) in LDs. The LD-binding Plin2 binds to both CE- and TAG-rich LDs, and although Plin2 is known to regulate degradation of TAG-rich LDs, its role for regulation of CE-rich LDs had been unclear. We showed that adrenal glands of Plin2-/- mice had significantly increased mass of CE-rich LDs in cortical cells, elevated cellular unesterified cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport. They also had high levels of phosphatidylglycerols, which paralleled the accumulation of ceroid-like structures; secretion of corticosterone induced by adrenocorticotropic hormone stimulation or starvation were, nonetheless, similar in Plin2+/+ and Plin2-/- mice. Our findings demonstrate an important role of Plin2 for regulation of CE-rich LDs and cellular cholesterol balance in the adrenal cortex.