Simulation of the Gαq /Phospholipase Cβ1 Signaling Pathway Returns Differentiated PC12 Cells to a Stem-like State

Abstract

Phospholipase Cβ1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters, and is found at high levels in mammalian neuronal tissue. Besides carrying out this key plasma membrane function, PLCβ1 has a cytosolic population that helps, in part, to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA-induced silencing (C3PO). Here, we show that down-regulating PLCβ1 or reducing its cytosolic population by activating Gαq to drive it to the plasma membrane, returns differentiated PC12 cells to an undifferentiated state. In this state, the cells return to a spherical morphology, resume proliferation and express the stem cell transcription factors nanog and Oct4. Similar changes are seen with C3PO down-regulation. This return to a stem-like state is accompanied by shifts in multiple miR populations, such as increased levels of rno-miR-21 and rno-miR-26a. Surprisingly, we find that de-differentiation can also be induced by extended stimulation of the Gαq. In this case, the neurites completely retract over a 10-minute period, and while levels of nanog remain unchanged, the levels of some miRs begin to return to their undifferentiated values. In complementary studies, we followed the real time hydrolysis of a fluorescent-tagged miR in cells where PLCβ1 or C3PO were down-regulated. These samples showed substantial differences in miR processing in cells both the undifferentiated and differentiated states. Taken together, our studies suggest that PLCβ1, through its ability to regulate C3PO and endogenous miR populations, plays a key role in mediating PC12 cell differentiation.2 succession, some occur over longer periods resulting in cells that may have a narrow or broad a distribution of differentiation. Reversal of differentiation often occurs in cells that have transformed to cancerous states [1]. Thus, the ability to control differentiation and induce dedifferentiation would greatly aid in optimizing cancer therapies and other therapies where inducing or reversing differentiation would be beneficial.In this study, we show that we can manipulate the differentiated state of PC12 cells, a cultured cell line that is commonly used as a model for neuronal cells, through the Gαq/PLCβ1 signaling pathway [2,3]. This pathway is initiated when neurotransmitters such as acetylcholine, serotonin, dopamine and melatonin bind to their specific transmembrane receptor. The ligandbound receptor then activates Gαq which in turn activates phospholipase Cβ, leading to an increase in intracellular calcium. There are four known families of PLCβ, and PLCβ1 is the most responsive to Gαq, and is highly expressed in neuronal cells, such as PC12.Aside from its classic role on the plasma membrane, PLCβ1 also has an atypical cytosolic population that binds to unique partners. Specifically, we have found that PLCβ1 binds to and inhibits component 3 promoter of RISC activity (C3PO), which promotes RNA-induced silencing by accelerating degradation of the passenger strand of the silencing ...

Repeated Activation of Gαq by neurotransmitters causes age-dependent accumulation of stress granules inC.elegans

“…Phase modulation FLIM measurements were performed on the dual-channel confocal fast FLIM (Alba version 5, ISS Inc.) using a two-photon titaniumsapphire laser and a Nikon Eclipse Ti-U inverted microscope as described (15). The lifetime of the laser was calibrated each time before experiments by measuring the lifetime of Atto 425 in water with a lifetime of 3.61 ns at 80 MHz.…”

“…This binding activates Gαq, which then activates PLCβ, setting off a series of events that result in increased intracellular calcium (12). We have previously found that in model cultural neuronal cells (PC12), Gαq activation causes irreversible retraction of neurites in the first 15 minutes of stimulation (14) and causes the cells to return to a stem-like state after 72 hrs post-stimulation (15). Alternately, in C.elegans, Gαq activation transiently ruptures neuronal connections and halts locomotion which then recovers after ~30 minutes presumably due to endogenous growth factors (14).…”