SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Post-transcriptional processes mediated by mRNA binding proteins represent important control points in the regulation of gene expression. Tristetraprolin (TTP) members are RNA binding and destabilizing proteins, characterized by a central specific tandem zinc finger and C-terminal, CNOT1 binding domain. We have characterized the single TTP member of the family in Dictyostelium discoideum TtpA. The Dictyostelium protein behaves identically to mammalian TTP family members in biochemical assays and promotes decay of mammalian transcripts that contain these motifs, when ectopically expressed in mammalian cells. We explored the physiological role of the single TTP family member (ttpA) expressed in Dictoystelium, using null, frame-shift, and point mutants. Null and frame-shift mutants exhibited slow growth and multinuclear phenotypes; cell phenotypes of a cysteine point mutant within the TZF domain, which prevent mRNA binding, and a carboxyl-terminal mutation, which removed the CNOT1 binding domain, were indistinguishable. All null, frame-shift, and point mutants exhibited reproducibly increased (>5x) expression of three specific transcripts, suggesting relief from TTP post-transcriptional repression. The carboxyl-terminal mutation exhibited an identical gene-expression phenotype, suggesting that this domain is primarily responsible for interactions with the CCR4/NOT mRNA decay complex. All target mRNAs contain the conventional UUA(UUUA)nUU TTP instability motifs in their 3UTRs, and a UUA(UUUA)nUU-containing 3-UTR was able to confer TtpA regulation to a heterologous mRNA; such regulation was lost upon mutation of the motif. A final striking finding is that all three of the TtpA target transcripts encode proteins of nearly identical membrane topology, characterized by signal peptides, large extracellular domains, single transmembrane domains, and short C-terminal cytoplasmic tails. These parallel topologies and related expression patterns suggest that the three transcripts are an example of an RNA regulon, in which ribonucleoprotein complexes coordinately regulate the production of functionally related proteins. When Dictyostelium are starved for nutrients, cells within a territorial space begin to 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. Although many components of these pathways are known (including receptors, G proteins, an adenylyl cyclase, phosphodiesterases, and protein kinases), they have not been biochemically analyzed in an integrated kinetic pathway. We now also identify new members that provide both feed-back and feed-forward processes.Through biochemical experiments, coupled with gene inactivation studies, we have proposed an integrated multi-component (>15 factor) pathway involving activations, inactivations (adaptation), and re-activations (re-sensitization) to generate cAMP oscillations. We build mathematical models to refine our experimental testing and explore additional component members. Cell-cell interaction and response 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 full-length DPF develop at low cell densities; cells deficient for DPF require higher cell density for development, compared to WT. DPF is synthesized as a larger precursor, single-pass transmembrane protein that is released by proteolytic cleavage and ectodomain shedding. We had proposed that the secreted 150 kDa protein was sufficient to regulate development non-autonomously, and that the TM/cytoplasmic domain of DPF possessed independent cell autonomous activity for cell-substratum adhesion and for cellular growth. We have now created vectors that allow expression of only the secreted or only the TM/cytoplasmic domain forms and are using them to test this model. We have also identified a sequence related protein with similar DPF properties and are testing potential pathway interactions among the two secreted proteins. We have also shown that DPF ectodomain cleavage is largely inhibited by depletion of calcium and are testing the role of calcium-dependent proteases(calpains)in this process. Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. Steroidogenic cells primarily store CEs in cytoplasmic lipid droplet (LD) organelles, 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 is unclear. To investigate the role of Plin2 in the regulation of CE-rich LDs, we performed histological and molecular characterization of adrenal glands from Plin2+/+ and Plin2-/- mice. Adrenal glands of Plin2-/- mice had significantly enlarged organ size, increased size and numbers of CE-rich LDs in cortical cells, elevated cellular unesterified cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport. Despite altered LD storage, mobilization of adrenal LDs and secretion of corticosterone induced by adrenocorticotropic hormone stimulation or starvation were similar in Plin2+/+ and Plin2-/- mice. Plin2-/- adrenals accumulated ceroid-like structures rich in multilamellar bodies in the adrenal cortex-medulla boundary, which increased with age, particularly in females. Finally, Plin2-/- mice displayed unexpectedly high levels of phosphatidylglycerols, which directly paralleled the accumulation of these ceroid-like structures. Our findings demonstrate an important role of Plin2 for regulation of CE-rich LDs and cellular cholesterol balance in the adrenal cortex. 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 isolated cardiomyocytes from adult Plin5+/+and Plin5-/- mice to study the role of Plin5 for fatty acid uptake, LD accumulation, fatty acid oxidation, and tolerance to hypoxia. Cardiomyocytes isolated from Plin5-/- mice cultured with oleic acid stored less LDs than Plin5+/+, but comparable levels to Plin5+/+ cardiomyocytes when adipose triglyceride lipase activity was inhibited. The ability to oxidize fatty acids into CO2 was similar between Plin5+/+ and Plin5-/- cardiomyocytes, but Plin5-/- cardiomyocytes had a transient increase in intracellular fatty acid oxidation intermediates. After pre-incubation with oleic acids, Plin5-/-cardiomyocytes retained a higher content of glycogen and showed improved tolerance to hypoxia compared to Plin5+/+. In isolated, perfused hearts, deletion of Plin5 had no important effect on ventricular pressures or infarct size after ischemia. Old Plin5-/- mice had reduced levels of cardiac triacylglycerides, increased heart weight, and apart from modest elevated expression of mRNAs for beta myosin heavy chain Myh7 and the fatty acid transporter Cd36, other genes involved in fatty acid oxidation, glycogen metabolism and glucose utilization were essentially unchanged by removal of Plin5. 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 of isolated cardiomyocytes has little importance for tolerance to acute hypoxia and ischemia, which contrasts the protective role for Plin5 in mouse models during myocardial ischemia, with a more complex metabolic interplay.