The Hippo pathway regulates organ size, regeneration, and cell growth by controlling the stability of the transcription factor, YAP (Yorkie in Drosophila). When there is tissue damage, YAP is activated allowing the restoration of homeostatic tissue size. The exact signals by which YAP is activated are still not fully understood, but its activation is known to affect both cell size and cell number. Here we used cultured cells to examine the coordinated regulation of cell size and number under the control of YAP. Our experiments in isogenic HEK293 cells reveal that YAP can affect cell size and number by independent circuits. Some of these effects are cell autonomous, such as proliferation, while others are mediated by secreted signals. In particular CYR61, a known secreted YAP target, is a non-cell autonomous mediator of cell survival, while another unidentified secreted factor controls cell size.
Muscle atrophy is a physiological response to disuse and malnutrition, but hibernating bears are largely resistant to this phenomenon. Unlike other mammals, they efficiently reabsorb amino acids from urine, periodically activate muscle contraction, and their adipocytes differentially responds to insulin. The contribution of myocytes to the reduced atrophy remains largely unknown. Here we show how metabolism and atrophy signaling are regulated in skeletal muscle of hibernating grizzly bear. Metabolic modeling of proteomic changes suggests an autonomous increase of non-essential amino acids (NEAA) in muscle and treatment of differentiated myoblasts with NEAA is sufficient to induce hypertrophy. Our comparison of gene expression in hibernation versus muscle atrophy identified several genes differentially regulated during hibernation, including Pdk4 and Serpinf1. Their trophic effects extend to myoblasts from non-hibernating species (including C. elegans), as documented by a knockdown approach. Together, these changes reflect evolutionary favored adaptations that, once translated to the clinics, could help improve atrophy treatment.
Translation of extracellular hormonal input into cellular responses is often mediated by repetitive increases in cytosolic free Ca 2+ concentration ([Ca 2+ ] c ). Amplitude, duration and frequency of these so-called [Ca 2+ ] c oscillations then carry information about the nature and concentration of the extracellular signalling molecule. At present, there are different hypotheses concerning the induction and control of these oscillations. Here, we investigated the role of agonist-induced receptor phosphorylation in this process using Chinese hamster ovary cells stably expressing a variant of the cholecystokinin 1 receptor (CCK1R) lacking the four consensus sites for protein kinase C (PKC) phosphorylation and deficient in CCK-induced receptor phosphorylation (CCK1R-mt cells). In the presence of cholecystokinin-(26-33)-peptide amide (CCK-8), these cells displayed Ca 2+ oscillations with a much more pronounced bursting dynamics rather than the dominant spiking dynamics observed in Chinese hamster ovary cells stably expressing the wild-type CCK1R. The bursting behaviour returned to predominantly spiking behaviour following removal of extracellular Ca 2+ , suggesting that CCK-8-induced, PKC-mediated CCK1R phosphorylation inhibits Ca 2+ influx across the plasma membrane. To gain mechanistic insight into the underlying mechanism we developed a mathematical model able to reproduce the experimental observations. From the model we conclude that binding of CCK-8 to the CCK1R leads to activation of PKC which subsequently phosphorylates the receptor to inhibit the receptormediated influx of Ca 2+ across the plasma membrane. Receptor-specific differences in this feedback mechanism may, at least in part, explain the observation that different agonists evoke [Ca 2+ ] c oscillations with different kinetics in the same cell type.
The Hippo pathway, in which changes at the cell surface and in the extracellular environment control the activity of a downstream transcription factor, known as YAP in mammalian cells and Yorkie in Drosophila, has recently taken center-stage as perhaps the most important pathway in metazoans for controlling organ size. In intact tissues YAP activity is inhibited and the organ does not overgrow. When the organ is damaged, YAP is active and necessary for growth and regeneration to occur. The exact process by which YAP drives organ and tissue growth is not fully understood, although it is known to affect both cell size and cell number. Since cell size and proliferation are highly interdependent in many cultured cell studies, we investigated the role of YAP in the simultaneous regulation of both cell size and number. Our experiments reveal that YAP controls both cell size and cell proliferation by independent circuits, and that it affects each process non-cell autonomously via extracellular mediators. We identify that CYR61, a known secreted YAP target, is the major regulator of the non-cell autonomous increase in cell number, but does not affect cell size. The molecular identity of the non-cell autonomously acting mediator of cell size is yet to be identified.
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