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

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.