OR ABSTRACT The second messenger cyclic AMP (cAMP) is closely linked with the control of adipogenesis, the de novo differentiation of fat cells. Its precise role, however, is disputed. Conflicting evidence in vitro and in mouse models suggests that cyclic AMP signals, which can be activated by many different physiological stimuli, are equally capable of inducing or inhibiting adipogenesis. Currently, it is unclear how the same signaling pathway can induce diametrically opposed effects upon differentiation. Cyclic AMP activates many effectors that all could influence a cell's decision to differentiate, and different mechanisms may prove dominant in different individual cells. Consequently, a population of precursors induced to differentiate will do so in an unsynchronized manner. In order to relate upstream signaling with eventual cell fate decisions, we must be able to make these measurements in the same cell. I hypothesize that anti- or pro-adipogenic environmental conditions are differentially encoded within the patterns of cAMP dynamics of individual cells, and these differential dynamics in turn induce or prevent adipogenesis. To test this hypothesis, I will express fluorescent sensors of cAMP-related activity in preadipocyte cell lines to measure live-cell signaling dynamics in differentiating cells. I will also express these sensors in a novel cell line expressing endogenous fluorescently- tagged PPAR?, the master regulator of adipogenesis. This tag allows the continuous tracking of differentiation state. With these tools I will determine which features of cAMP signaling are most closely associated with cells that are induced or blocked from differentiation. I will expose reporter cells to a variety of adipogenic environments, altering both cAMP stimulus type and the timing of its application as cells undergo differentiation, measuring the resultant induction of cAMP activation, and the eventual fate of thousands of cells in the course of a single experiment. Next, I will develop a methodology for understanding how interplay between cAMP effectors determines cell fate. While several mechanisms have been shown to allow cAMP to enable or prevent adipogenesis, the relative importance and roles of these candidates are poorly understood. I will evaluate whether these mechanisms are differentially regulated by different cAMP signaling patterns, and how they work together to control cell fate. Finally, I will test how cAMP activity controls differentiation in different adipocyte precursors from different fat depots. I have generated a novel mouse reporter, also expressing fluorescently-tagged PPAR?. I will isolate adipocyte precursors from different fat depots of these mice, and will differentiate them using a panel of cAMP stimuli, testing whether the same stimulus can be inhibitory with some precursor types, but induce adipogenesis in others. With this methodology, I can elucidate how different stimuli are translated into common patterns of cAMP activity within a cell, which in turn either induce or prevent differentiation. This dynamic ?code?, and the mechanisms that interpret it, provides a rational basis for governing adipogenesis by selectively altering, rather than blocking, this factor.
This work is aimed at resolving the role of cyclic AMP (cAMP), a signaling system with far-ranging effects in many cell types, on the process of adipogenesis, the differentiation of new fat cells. Cyclic AMP's role is disputed; cells can use this signal to either efficiently block or be essential in inducing differentiation. By understanding how cells use this pathway to decide their fate, I can identify new ways of encouraging healthy fat cell behavior, helping to treat metabolic disease and diabetes.
| Kovary, Kyle M; Taylor, Brooks; Zhao, Michael L et al. (2018) Expression variation and covariation impair analog and enable binary signaling control. Mol Syst Biol 14:e7997 |
