Phospholipase Cβ-TRAX Association Is Required for PC12 Cell Differentiation*

When treated with nerve growth factor, PC12 cells will differentiate over the course of several days. Here, we have followed changes during differentiation in the cellular levels of phosphoinositide-specific phospholipase Cβ (PLCβ) and its activator, Gαq, which together mediate Ca2+ release. We also followed changes in the level of the novel PLCβ binding partner TRAX (translin-associated factor X), which promotes RNA-induced gene silencing. We find that the level of PLCβ increases 4-fold within 24 h, whereas Gαq increases only 1.4-fold, and this increase occurs ∼24 h later than PLCβ. Alternately, the level of TRAX remains constant over the 72 h tested. When PLCβ1 or TRAX is down-regulated, differentiation does not occur. The impact of PLCβ on differentiation appears independent of Gαq as down-regulating Gαq at constant PLCβ does not affect differentiation. Förster resonance energy transfer studies after PLCβ association with its partners indicate that PLCβ induced soon after nerve growth factor treatment associates with TRAX rather than Gαq. Functional measurements of Ca2+ signals to assess the activity of PLCβ-Gαq complexes and measurements of the reversal of siRNA(GAPDH) to assess the activity of PLCβ-TRAX complexes additionally suggest that the newly synthesized PLCβ associates with TRAX to impact RNA-induced silencing. Taken together, our studies show that PLCβ, through its ability to bind TRAX and reverse RNA silencing of specific genes, plays a key role in switching PC12 cells to their differentiated state.

Differentiation of cells to a neuronal phenotype is a complex process involving a series of genetic and morphological changes. PC12 cells, which are derived from a pheochromocytoma of the rat adrenal medulla, have an embryonic origin from the neural crest (1) and are often used as model systems to study neural differentiation. PC12 cells are highly proliferative in their undifferentiated state. When treated with nerve growth factor (NGF), proliferation ceases and differentiation begins as seen by the onset of neurite growth as well as accumulation of Ca 2ϩ vesicles (2). NGF binds to TrkA growth factor receptors (3,4) leading to changes in transcription of a number of pro-teins that are associated with the differentiated state (5). Differentiation of PC12 cells is generally considered complete when the length of the neurites is three-four times the length of the cell body. One set of proteins that are associated with the differentiated state are those that control acetylcholine, serotonin, and dopamine pathways that lead to increased intracellular calcium (i.e. members of the G-protein/phospholipase C␤ signaling pathway) (6).
Our laboratory has actively studied the G␣ q /PLC␤ 2 signaling system (e.g. Refs. [7][8][9]. This pathway is activated by many types of neurotransmitters and hormones such as acetylcholine, dopamine, angiotensin II, bradykinin, etc. (10,11). Binding of these ligands to their specific G protein-coupled receptor results in the activation of G␣ q (GTP) that in turn stimulates the ability of PLC␤ to catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate, which ultimately results in the release of intracellular Ca 2ϩ from stores in the endoplasmic reticulum. PLC␤ (␤1-4) enzymes vary in their tissue distribution and their ability to be activated by G␣ q. This study focuses on PLC␤1 as it is strongly activated by G␣ q , is highly expressed in neural tissue, and can shuttle between the plasma membrane, cytosol, and nucleus (12)(13)(14)(15).
In PC12 cells PLC␤1 is primarily found on the plasma membrane in a complex with G␣ q , in addition to the cytoplasm and the nucleus (16). Recently, we made the surprising discovery that cytosolic PLC␤ binds to the promoter of RNA-induced silencing, C3PO (17). Additionally, we found that the association of PLC␤ to C3PO reverses the RNA-induced silencing of specific genes, such as GAPDH (8). Further studies using purified proteins suggested that this specificity results from the ability of PLC␤ to inhibit the activity of C3PO toward substrates that are rapidly hydrolyzed (18). Because the binding region of C3PO on PLC␤ enzymes directly overlaps with the G␣ q binding site, C3PO competes with G␣ q for PLC␤. Thus, whereas C3PO does not affect the basal activity of PLC␤, it prevents its activation by G␣ q (8,17).
C3PO crystallizes as an octamer containing 6 molecules of the single-stranded DNA/RNA-binding protein translin and 2 molecules of the nuclease translin-associated factor X (TRAX) (19). The expression of translin and TRAX are closely linked (20). Previous studies from our laboratory have shown that PLC␤ binds to an external site on either one or both of the TRAX subunits to inhibit TRAX activity (18). In addition to playing a role in tRNA processing, C3PO has also been shown to promote RNA-induced gene silencing by promoting the degradation of the passenger strand of duplex silencing RNA to allow hybridization of the target mRNA to the guide strand of the interfering RNA (21).
Although we have gathered ample evidence that PLC␤ binds to C3PO, the biological significance of this interaction has remained unclear. We have evidence that PLC␤ can shuttle between G␣ q on the plasma membrane and C3PO in the cytoplasm (22). Activation of G␣ q reduces the association between PLC␤ to C3PO, whereas treatment of cells with siRNA attenuates PLC␤-mediated Ca 2ϩ signals (23), leading to the idea that the impact of PLC␤1 on RNA silencing may be related to the relative levels of the two PLC␤ binding partners C3PO and G␣ q . In this study we show that the initial stages of differentiation of PC12 cells depends on increased association between PLC␤ and C3PO rather than between PLC␤ and G␣ q . Thus, the ability of PLC␤ to modulate RNA silencing through its association with C3PO demonstrates that this unexpected secondary function of PLC␤ plays a role in certain physiological processes.

Results
Differentiation Induces Large Changes in the Cellular Levels of PLC␤1 and G␣ q at Different Rates but Not TRAX-In their undifferentiated state, PC12 cells have a spherical morphology. Upon NGF treatment, the cells begin to sprout neurites within a few hours. Differentiation is generally considered complete when the length of the neurites is three times the length of the cell body. Under our conditions, we find that the ratio of the length of the neurites versus the length of the body reaches 3.0 ϳ36 h after NGF treatment under our conditions.
We followed the changes in expression of PLC␤1, TRAX, and G␣ q as a function of time after NFG is added to PC12 cells to initiate differentiation. Interestingly, we found a marked increase in the level of PLC␤1 (i.e. 2.5-2.7-fold) in first 24 h. This increase peaks to 4-fold at 48 h (Fig. 1A) and decreases thereafter. Although G␣ q also showed a large increase in expression with differentiation (1.6-fold), the onset of this increase was delayed 24 h relative to PLC␤ (Fig. 1A). The levels of G␣ q began to decline after 48 h but still remained higher than in the undifferentiated state (Fig. 1A). In contrast to PLC␤1 and FIGURE 1. A, changes in the cellular levels of PLC␤1, G␣ q , and TRAX in PC12 cells after the addition of NGF as determined by Western blotting where protein levels were normalized to loading control (␤-actin) and where the data were compiled from six independent experiments. Changes in TRAX were found to not be significant, but changes in PLC␤1 and G␣ q at 48 and 72 h were significant compared with undifferentiated conditions (p Ͻ 0.001). Also shown on the right is a sample Western blot where UN refers to undifferentiated PC12 cells. B, increases in the membrane-associated PLC␤1 in PC12 cells where membrane fractions were isolated as described under "Experimental Procedures," and the amount of enzyme was divided by the total cellular level of PLC␤1. The data were compiled from three separate experiments, and a sample Western blot is shown. All points on the graph are statistically different (p Ͻ 0.001).
G␣ q , no changes in the level of TRAX, and in turn C3PO (20), were observed over 72 h after NGF treatment (Fig. 1A).
Although we have only observed G␣ q on the plasma membrane in PC12 cells (16), PLC␤ has been identified in the nucleus, cytoplasm, and the plasma membrane (15,16). We wanted to determine whether the increase in PLC␤ that occurs with differentiation localizes to the plasma membrane or other compartments. These studies were done by assessing the amount of endogenous enzyme found in the whole cell by Western blotting and comparing it to the level found in the membrane fractions. We note that PLC␤1 in the cytosol is too dilute to accurately quantify. We find that 24 h after NGF treatment, when PLC␤ production is highest, the level of membrane-bound PLC␤ corresponds to approximately half the newly produced PLC␤ (Fig. 1B). However, at 48 h, when G␣ q levels peak, almost all of the enzyme is associated with the membrane fraction. These results suggest that half of the newly synthesized enzyme remains in the cytosol until enough available G␣ q is made.
Down-regulation of PLC␤ and TRAX, but Not G␣ q , Prevents Differentiation-Based on the large increases in the levels of PLC␤ and G␣ q with differentiation, we tested the idea that eliminating the expression of PLC␤ would prevent differentiation. PLC␤ was down-regulated by ϳ80% using siRNA 24 h before treatment with NGF. We find that cells transfected with siRNA (PLC␤1) did not differentiate as assessed by a lack of neurite growth over 72 h of NGF treatment (Fig. 2, A and B).
To determine whether the lack of differentiation caused by PLC␤1 down-regulation is due to its ability to mediate G protein signals, we repeated the study down-regulating G␣ q . Because down-regulating G␣ q also reduced the expression of PLC␤1 (Fig. 2C), we cotransfected PLC␤1 with siRNA(G␣ q ) to maintain a constant level of PLC␤ (see Fig. 2C) and measured differentiation. We find that the cells differentiated normally (Fig. 2, A and B), suggesting that G␣ q is not involved in differentiation.
We then tested the role of TRAX on differentiation and note that down-regulation of TRAX down-regulates translin and hence C3PO (see Ref. 20). We also note that down-regulating TRAX does not affect PLC␤1 expression (Fig. 2C). Similar to the results seen for PLC␤1, down-regulating TRAX blocks PC12 cell differentiation (Fig. 2, A and B). Taken together, these results suggest that PLC␤ and C3PO, either together or in isolation, play a key role in PC12 cell differentiation.
PLC␤ Changes Its Association with Its G␣ q and TRAX Binding Partners during Differentiation-To test whether the association between TRAX and PLC␤1 was one of the factors that prevented PC12 cells differentiation, we followed the association of PLC␤ to TRAX and also to G␣ q after NGF treatment using Főrster resonance energy transfer (FRET) (see Ref. 16). We first examined changes in the relative number of eCFP-G␣ q /eYFP-PLC␤1 complexes, which based on the results above, are not expected to affect differentiation. We note that FRET is a ratiometric quantity, and in order to obtain accurate FRET values we viewed cells in which the donor and acceptor intensities were within a factor of 10, which should not bias the results (see Ref. 25). Measurements were limited to the plasma membrane region where G␣ q is localized, and this was verified by the absence of PLC␤-G␣ q FRET in the cytoplasm. The results are shown in Fig. 3, A and B. We find little change in FRET from 0 to 24 h after the addition of NGF, suggesting that the amount of PLC␤-G␣ q FRET is unchanged. This result suggests that the increased cellular amount of PLC␤ that is produced during this time does not result in an increase in the number of PLC␤-G␣ q complexes. At 48 h, where the level of G␣ q sharply rises, we find a large population of fluorescent-tagged proteins is not associated with PLC␤, which may reflect initial association of the newly synthesized G␣ q still being trafficked to the plasma membrane as noted by the observation of the protein in cytosolic endosomes or not in optimal orientation for productive FRET. However, as the levels of G␣ q on the plasma membrane rise at 72 h, the level of FRET reaches a higher value consistent with previous studies (8).
We used the same FRET approach to monitor changes in the association between PLC␤ and TRAX with differentiation in the cytosol where the majority of the complexes have been identified (17,23). Upon NGF treatment, we observed an ϳ2-fold increase in the amount of FRET (14.2 Ϯ 0.08 to 28.2 Ϯ 0.09% (p Ͻ 0.001) when normalized to positive and negative controls) in the first 12 h and a reduction in FRET thereafter (Fig. 3C). We note that the increase in association at 12 h rather than 24 reflects the higher initial level of PLC␤ due to overexpression of the fluorescent construct (see Fig.  3D).
Changes in the Relative Levels of G␣ q and TRAX with Differentiation Affect Ca 2ϩ Signals and RISC Activity-We measured changes in Ca 2ϩ responses through the G␣ q -PLC␤ pathway in undifferentiated cells and after NGF treatment. These studies were carried out by stimulating a suspension of cells (10 6 cells/ ml) loaded with a fluorescent Ca 2ϩ indicator (Fura-2) with carbachol. We find that the amount of Ca 2ϩ released at 0 and 24 h after NGF treatment were similar to each other and lower than the amount released at 48 h (Fig. 4). This behavior parallels the increase in G␣ q levels with differentiation.
We have previously shown that overexpression of PLC␤1 can reverse down-regulation of specific proteins through its association with C3PO (8). We tested reversal in siRNA down-regulation of GAPDH and compared these results to an siRNA for a message that PLC␤1 is not able to reverse (siRNA(Hsp90)). The results (Fig. 5) show that at 24 h after NGF treatment, when the PLC␤1 levels are high but G␣ q levels are low, the ability of PLC␤1 to reverse down-regulation of GAPDH by siRNA is significantly less efficient, whereas Hsp90 levels are unaffected. However, when G␣ q levels rise to drive PLC␤ binding, as seen by an increased calcium response, the ability of PLC␤ to reverse C3PO activity is greatly reduced. These studies show that higher PLC␤ levels impacts on RISC activity through its association with TRAX.

Discussion
In this study we show that PLC␤ and TRAX are required for PC12 cell differentiation. This finding demonstrates that the ability of PLC␤ to inhibit TRAX may play a key biological role in specific cellular functions. Previously, PLC␤ enzymes were not thought to impact differentiation. Instead, changes in phosphatidylinositide signals during differentiation were found to be initiated by PLC␥ subsequent to activation of TrkA receptors upon NGF treatment (see Ref. 26). It is notable that the Ca 2ϩ released by PLC␥ activity in turn stimulates the highly active PLC␦ synergizing the calcium signal. The very high specific activity of PLC␦ as compared with PLC␤ upon NFG treatment, especially at low G␣ q concentrations, suggests that PLC␤ would contribute little, if any, to phosphatidylinositol 4,5 bisphosphate hydrolysis and the Ca 2ϩ levels. This observation supports the idea that PLC␤'s traditional role as the main effector of G␣ q does not impact differentiation.
It has been established that PLC␤1 levels correlate closely with neuronal differentiation in both cat and rat somatosensory cortex (27), establishing a role of PLC␤1 in brain development. Therefore, it is not surprising that large increases of both G␣ q and PLC␤ levels are associated with PC12 cell differentiation as the cells establish sensory Ca 2ϩ signaling pathways. However, it was surprising to find that a very large increase in PLC␤ expression occurs before the onset of increased G␣ q levels. This finding correlates well with the idea that the phosphoinositide lipid signaling function of PLC␤ does not appear to play a role in the initial stages of differentiation, as indicated by the lack of effect of G␣ q down-regulation.
Our studies suggest that the increase in PLC␤ levels and the relative changes in its two binding partners coordinate to allow PC12 cell differentiation. PLC␤ levels increased 24 h before G␣ q , and the newly synthesized enzyme interacts with TRAX as observed by FRET studies showing an increase in the number of PLC␤-TRAX complexes. However, this increase in FRET reverses at 48 h, which correlates to the increased level of G␣ q . Localization studies at these later times show a large increase in the membrane-bound population of PLC␤ consistent with the idea that it is being driven to the membrane and away from TRAX as G␣ q is synthesized and more PLC␤1 binding sites become available. Although we expected to observe an increase in FRET between G␣ q and PLC␤ at 48 h, we instead find a strong contribution of weakly transferring species. This low FRET population may be due to either uncomplexed protein or proteins in an orientation where the probability of FRET is reduced. Previous diffusion studies of PLC␤ in PC12 cells 48 h after NGF treatment show a very limited mobility (16), indicating that PLC␤ is associated with slower moving species (presumably G␣ q ). Thus, at 48 h after NGF treatment PLC␤ may be driven to the membrane by G␣ q but not in close enough proximity to the protein or in a favorable orientation for FRET. In any event, our results suggest that the delayed synthesis of new G␣ q competes with TRAX for PLC␤ until the final differentiated state is reached. The idea that differentiation proceeds through changes in the number of PLC␤-TRAX versus PLC␤-G␣ q complexes can be seen in studies where we overexpressed G␣ q to drive PLC␤ from TRAX, and we find that differentiation no longer occurs.
Previous studies have suggested that PLC␤ is not involved in PC12 cell differentiation (28). However, in those studies, the importance of PLC␤ was assessed by over-expressing the enzyme, and here we also show that over-expressing PLC␤ does not affect differentiation. Instead, we find that a minimal amount of PLC␤ is needed to interact with TRAX and drive differentiation. This idea is supported by the absolute need for TRAX in the differentiation process. The observation that excess PLC␤ neither diminishes nor promotes this process suggests that there is a limited number of TRAX binding sites for PLC␤.
C3PO has been reported to play a role in tRNA processing and promote mRNA degradation by the RNA-induced silencing complex (17,19,21). Like PLC␤1, TRAX also is associated with neuronal development (24). Thus, the finding that TRAX, which is responsible for C3PO nuclease activity, is required for PC12 cell differentiation was not unexpected. We have previously shown that PLC␤ binds to the TRAX subunits of C3PO and inhibits its activity (18). Thus, it is possible that in the undifferentiated state, C3PO helps to transiently silence specific genes that interfere with the progression of cells into the differentiated state. Our studies showing the reversal of siRNA(GAPDH) silencing at 24 h, when the levels of G␣ q are low and PLC␤ binding to TRAX is promoted, correlate well with this idea. After this initial period, as more G␣ q is synthesized, PLC␤ dissociates from TRAX and presumably moves to the plasma membrane to carry out its traditional role in G protein signaling as differentiation is completed. In this way, PLC␤ acts as an off/on switch to inhibit the transcription of genes that interfere with differentiation. We are in the process of identifying the specific genes.

Experimental Procedures
PC12 Cell Culture and Differentiation-PC12 cells were purchased from American Tissue Cell Culture and cultured in DMEM (Invitrogen) with 10% horse serum from PAA (Ontario, Canada), 5% FBS (Atlanta Biological, Atlanta, GA), and 1% pen- icillin/streptomycin. Cells were incubated in 37°C, 5% CO 2 , and 95% humidity. Differentiation was carried out in a medium of 1% horse serum, 0.5% FBS, 0.5% penicillin/streptomycin and was initiated by the addition of nerve growth factor (NGF 7S) from Sigma. Medium was changed every 24 h.
Western Blotting-Samples were placed in 6-well plates and collected in 250 l of lysis buffer that included Nonidet P-40 and protease inhibitors. After SDS-PAGE electrophoresis, protein bands were transfer to nitrocellulose membrane (Bio-Rad). Primary antibodies to PLC␤1 (D-8), G␣ q (E-17), and TRAX (E-11) were from Santa Cruz (Dallas, TX). Antibodies for ␤-actin and GAPDH from were from Abcam. Membranes were treated with antibodies diluted 1:1000 in 0.5% dry milk and washed 3 times for 5 min before applying secondary antibiotic (anti-mouse or anti-goat from Santa Cruz) at a concentration of 1:500. Membranes were washed 3 times for 5 min before imaging on a Bio-Rad imager to determine the band intensities. Bands were measured at several sensitivities to ensure the intensities were in a linear range. Data were analyzed using Image J in grayscale plot profile. Bands were normalized to loading control.
Transfection-Plasmid transfections were accomplished using Lipofectamine 3000 (Invitrogen) as recommended by the manufacturer. Transfection of siRNAs was also carried out using Lipofectamine 3000.
Transfection for FRET Studies-Cells were seeded in 35-ml dishes. At 60% confluence cells were transfected using Lipofectamine 3000 with 1 l of DNA of the target protein according to the manufacturer's protocol. Cells were given 3 days to express the fluorescent proteins. Four separate samples were prepared: empty vectors, donor alone, acceptor alone, and both donor and acceptor.
Plasma Membrane Isolation-1 ϫ 10 7 cells were collected and homogenized manually. Trypan blue was used to check the quality of homogenization. Lysed cells were centrifuged at 4°C for 3 min at 4000 rpm to isolate nuclei. The supernatant was collected and subjected to ultracentrifugation at 45,000 rpm for 45 min to separate the cell membrane.
Confocal Imaging-Cells were seeded in poly-D-lysinecoated glass-covered dishes from Mat-Tek. Images were acquired on an Olympus Fluorview 1000 confocal microscope and Zeiss 510 Meta confocal microscope. Data were analyzed using Olympus Fluoview 1000 software and Image J software.
Calcium Measurements-Single cell calcium measurements were carried out by labeling the cells with calcium green (Thermo Fisher Scientific) in incubated in HBSS for 45 min and washed twice with HBSS before imaging. Ensemble calcium measurements were carried out by preparing cells in 100-ml dishes, washing with HBSS, harvesting, and labeling with Fura-2 (Invitrogen) for 45 min in HBSS with 1% BSA and 5 mM glucose. Cells were washed twice, adjusted to a count of 1 ϫ 10 6 cells/ml, and placed in 1-ml cuvettes. Cells were stimulated with 1 M carbachol. Calcium range was determined by disrupting cells by 10% Triton X-100 and by 200 mM EDTA to chelate calcium.
Statistical Analysis-Data were analyzed using Sigma Plot 11 statistical packages that included Student's t test and one way analysis of variance.
Author Contributions-O. G. carried out the experimental work and analysis. S. S. helped with experimental design, data analysis, and the writing of this paper.