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Phospholipase C-η2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCη2 activity is triggered by intracellular calcium mobilization and likely serves to amplify Ca2+ signals by stimulating further Ca2+ release from Ins(1,4,5)P3-sensitive stores. The role of PLCη2 in neuritogenesis was assessed during retinoic acid (RA)-induced Neuro2A cell differentiation. PLCη2 expression increased two-fold during a 4-day differentiation period. Stable expression of PLCη2-targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA-treatment. Furthermore, RA response element activation was perturbed by PLCη2 knockdown. Using a bacterial two-hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCη2. Immunostaining of PLCη2 revealed significant co-localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA-induced phosphorylation of LIMK1 and cAMP-responsive element-binding protein was reduced in PLCη2 knock-down cells. The phosphoinositide-binding properties of the PLCη2 PH domain, assessed using a FRET-based assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P3. Immunostaining of PLCη2 together with PtdIns(3,4,5)P3 in the Neuro2A cells revealed a high degree of co-localization, indicating that PtdIns(3,4,5)P3 levels in cellular compartments are likely to be important for the spatial control of PLCη2 signaling.
Phospholipase C-η (PLCη) enzymes are a recently identified class of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-hydrolyzing enzyme found in mammalian cells (Hwang et al. 2005; Nakahara et al. 2005; Stewart et al. 2005; Zhou et al. 2005). This class consists of two members, PLCη1 and PLCη2. Like other mammalian PLCs, their activity leads to the production of the second messengers Ins(1,4,5)P3 and 1,2-diacylyglycerol (DAG), which trigger the release of Ca2+ from intracellular stores and the activation of protein kinase C isoforms, respectively.
Phospholipase C-η2 is most abundant in the brain and is found within the hippocampus, habenula, olfactory bulb, cerebellum, and throughout the cerebral cortex (Nakahara et al. 2005; Kanemaru et al. 2010). Activation of PLCη2 can be triggered either by intracellular Ca2+ immobilization (Popovics et al. 2011) or by Gβγ signaling (Zhou et al. 2005, 2008). Considering its sensitivity of toward Ca2+, it is thought that PLCη2 may act synergistically with other PLCs or Ca2+ activated processes in neurons (Popovics and Stewart 2012). When transiently expressed in COS7 cells, PLCη2 associates with plasma and organelle membranes via its PH domain (Popovics et al. 2011). However, the specificity of this domain toward particular phospholipids, which is likely to control its cellular location, remains unknown.
Although a role in the amplification of PLCβ signals has recently been demonstrated for PLCη1 (Kim et al. 2011), a functional role has yet to be identified for PLCη2. Nakahara et al. previously reported that levels of PLCη2 protein increase in the brains of mice during the first few weeks after birth (Nakahara et al. 2005). Similarly, the retina expresses high levels of PLCη2 which also elevate from birth (Kanemaru et al. 2010). Its expression profile hints that this enzyme might be involved in processes linked to neuronal differentiation or maturation. In this study, we examined the importance of PLCη2 during retinoic acid (RA)-induced differentiation of Neuro2A cells. This system was chosen as it provides a well-defined cellular model of neuronal differentiation that has been used to characterize the importance of various factors in this process (Pignatelli et al. 1999; Carter et al. 2003; Bryan et al. 2006; Kouchi et al. 2011), and these cells are known to express endogenous PLCη2 (Kim et al. 2011). Stable shRNA-mediated knockdown of PLCη2 expression in these cells revealed this enzyme to be important for RA-stimulated neurite outgrowth. Reductions in PLCη2 expression in isolated clones led to reductions in RA signaling and LIM kinase-1 (LIMK1) phosphorylation. A bacterial two-hybrid screen identified LIMK1 as a putative interaction partner for PLCη2. Furthermore, it was found that PLCη2 co-localizes with LIMK1 within the nucleus and growing neurites in Neuro2A cells and that PLCη2 associates with intracellular PtdIns(3,4,5)P3 pools via its PH domain.
- Top of page
- Materials and methods
- Supporting Information
Neuro2A cells represent an excellent model for the study of neuronal differentiation and were used to assess the role of PLCη2 in this process. PLCη2 protein expression was found to increase in Neuro2A cells during retinoic acid induced differentiation. The involvement of PLCη2 in this process was further indicated by an observed reduction in neurite outgrowth in shRNA-mediated PLCη2 knock-down cells (shRNAPLCη2-1 and shRNAPLCη2-2 lines). Both the percentage of differentiated cells and the total neurite growth were found to be reduced. The extent of this phenotypic effect largely mirrored the degree by which PLCη2 expression was decreased in the PLCη2 knock-down cells, highly indicative of a specific role for PLCη2 in this process. Differentiating neurons are known to exhibit spontaneous Ca2+ spikes and waves (Gu and Spitzer 1995) and, because of the high Ca2+ sensitivity of PLCη2, it is possible that the enzyme plays an important role in the generation of these signals and/or their translation into downstream effects. This is supported by the observation that inositol phosphate release is reduced in PLCη2 knock-down cells, relative to control cells, following treatment with the Ca2+ ionophore, A23187. It should be acknowledged that most of the inositol phosphate release triggered by this agent seems to be directed by PLCη2 with other PLCs having little residual effect. This is likely down to that fact that PLCη enzymes are more sensitive to Ca2+ in comparison to other isotypes likely to be present (such as PLCδ enzymes). PLCη1 has been shown to amplify PLCβ signals in Neuro2A cells following Ca2+ release (Kim et al. 2011). Whether PLCη1 and PLCη2 signals are interdependent or not is still to be established but it is possible that lowering PLCη2 protein level would greatly impact upon PLCη signaling as a whole.
To determine whether PLCη2 regulates transcriptional activity we examined its influence in the control of RAR-directed gene expression. Of the three known RAR isoforms, only RARα was found to be present in Neuro2A cells. The mRNA level corresponding to expression of this isoform was found to be similar in control and PLCη2 knock-down cells. However, PLCη2 knock-down had a substantial effect on basal and RA-stimulated retinoid signaling. This likely contributes to the phenotype associated with the PLCη2 knock-down cells as neuronal differentiation is, as one would expect, associated with a substantial change in gene expression (Gurok et al. 2004). The precise mechanism(s) by which PLCη2 exerts this effect is not clear. Promoters containing RAREs have been shown to be activated by downstream regulatory element antagonist modulator, a Ca2+-effector protein (Scsucova et al. 2005). It is possible that the absence of sufficient levels of PLCη2 in the knock-down cells negatively influences cytosolic Ca2+-dynamics such that activation of downstream regulatory element antagonist modulator is compromised. PLCη2 was found to be present in the nuclei of both undifferentiated and differentiated Neuro2A cells. Other nuclear PLC enzymes are known to influence gene expression. A prime example is PLCβ1, which is present in nuclear speckles; the location of several transcription-regulating molecules (Martelli et al. 1992). PLCβ1 is involved in the regulation of c-Jun and AP1 promoter-binding in differentiating myogenic cells (Ramazzotti et al. 2008). As with cytosolic PLC enzymes, the nuclear enzymes can activate protein kinase C (PKC) (α-isoform) via the production of DAG. PKCα has been shown to phosphorylate lamin B1 to trigger the breakdown of the nuclear lamina for mitotic division in murine erythroleukemia cells (Fiume et al. 2009). The consequent action of PLC enzymes in the nucleus decrease PtdIns(4,5)P2 levels, which are important for chromatin remodeling (Zhao et al. 1998). It has also been proposed that cells may possess Ins(1,4,5)P3 receptors on the inner nuclear membrane that allow movement of Ca2+ to the nucleoplasm (Klein and Malviya 2008). Despite this, the precise roles PLC enzymes play in regulating gene expression and nuclear Ca2+ dynamics are still far from understood.
A putative interaction between PLCη2 and LIMK1 was identified using a bacterial two-hybrid screen. We attempted to confirm this interaction in Neuro2A cells using a co-immunoprecipitation approach but were unsuccessful (data not shown). Nevertheless, LIMK1 and PLCη2 do co-localize in both undifferentiated and differentiated Neuro2A cells. This implies that their distribution allows the interaction of these proteins. LIMK1 and CREB phosphorylation was significantly reduced in the PLCη2 knock-down cells, suggesting that PLCη2 regulates their activity. PLCη2 may potentially influence LIMK1 (and CREB) phosphorylation in two ways. First, it remains known that the phosphorylation of LIMK1 and CREB is regulated by Ca2+ via the Ca2+/calmodulin kinase IV pathway (Matthews et al. 1994; Takemura et al. 2009), thus phosphorylation of both proteins may be altered because of aberrations in Ca2+-signaling in the PLCη2 knock-down cells. Second, it is also possible that PLCη2 and LIMK1 interact directly in such a manner as to regulate activation of LIMK1. It is also possible that a PLCη2-LIMK1 interaction could simply serve such that PLCη2 is “on hand” to modulate LIMK1 activation by Ca2+/calmodulin kinase IV. By whichever mechanism, both PLCη2 and LIMK1 are present in growing neurites. Reduced phosphorylation of LIMK1 (as observed in the PLCη2 knock-down cells) is likely to influence phosphorylation of cofilin. Once phosphorylated, cofilin acts to alter the F-actin/G-actin ratio, a process which is important for neurite outgrowth (Yang et al. 1998, 2004). Aberrations in actin dynamics were observed by phalloidin-tetramethylrhodamine B isothiocyanate staining of shRNAPLCη2-1 and shRNAPLCη2-2 cells. In these cells, a punctate distribution of F-actin was observed with multiple projections and reduced outgrowth compared to control cells. Interestingly, Endo et al. (2007) previously found that down-regulation of LIM kinase activity in PC12 cells significantly reduces neuronal outgrowth. Although the authors did not mention it, the number of projections also appeared to be increased in these cells. Potentially, the observed phenotype of PLCη2 knock-down cells could be caused, at least in part, by an increase in cofilin activity and a consequent reduction in the polymerization rate of actin. Such effects are likely to be further compounded in PLCη2 knock-down cells by reduced expression of RARα-regulated genes involved in the differentiation process (as summarized in Fig. 7). In addition, significant staining of LIMK1 was found in the nucleus of the Neuro2A cells. Nuclear translocation LIMK1 has previously been suggested to be a process important for the control of actin dynamics (Yokoo et al. 2003). It is also possible that nuclear LIMK1 activity contributes to the regulation of genes involved in neuritogenesis.
Figure 7. Summary of pathways by which Phospholipase C (PLC)-η2 may regulate neuronal differentiation. Upon PLCη2 activation, the level of the substrate PtdIns(4,5)P2 will decrease with subsequent elevations in Ins(1,4,5)P3 and 1,2-diacylyglycerol (DAG). Ins(1,4,5)P3 triggers Ca2+-release from the endoplasmic reticulum to activate LIM kinase-1 (LIMK1) and subsequently cAMP-responsive element-binding protein (CREB) through stimulation of CaMKIV activity. PLCη2 could also potentially activate LIMK1 by direct association. Activated CREB initiates gene transcription. Activated LIMK1 also inhibits actin depolymerization leading to the assembly of actin filaments necessary for neuronal differentiation. Changes in the nuclear DAG and PtdIns(4,5)P2 levels may also affect the transcriptional activity through DAG-PKC or PtdIns(4,5)P2-chromatin interactions. Abbreviations: PLCη2: Phospholipase C-η2, InsP3: Inositol 1,4,5-trisphosphate, PtdIns(4,5)P2: Phosphatidylinositol 4,5-bisphosphate, DAG: diacylglycerol, CaMKIV: Ca2+/Calmodulin-dependent protein kinase IV, LIMK1: LIM domain kinase 1, cAMP response element-binding protein, PKC: Protein kinase C.
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The PH domain of PLCη2 has previously been shown to be responsible for association of the enzyme to intracellular membranes (Nakahara et al. 2005; Popovics et al. 2011). A GST-tagged PLCη2 PH domain was synthesized and its ability to bind a range of lipids and phosphoinositides was examined. The PLCη2 PH domain bound preferentially to PtdIns(3,4,5)P3. Furthermore, PLCη2 was found to co-localize with PtdIns(3,4,5)P3 in the nucleus and to some extent at the cell membrane in Neuro2A cells, suggesting that it predominantly interacts with this lipid in vivo. Neuronal cells have previously been reported to possess high levels of PtdIns(3,4,5)P3 levels in the nucleus (Neri et al. 1999; Kwon et al. 2010). This likely explains why PLCη2 is present in the nucleus of Neuro2A cells and why its cellular distribution differs in other non-neuronal cell types (Popovics et al. 2011). It is important to point out that the PLCη2 PH domain was also able to bind PtdIns(4,5)P2, although with a somewhat lower affinity. However, co-expression of GFP-PLCη2-PH with mCherry-tagged PLCδ1-PH domain in Neuro2A cells revealed the cellular locations of these proteins to be distinct. The PH domain of PLCδ1 is known to associate with cellular PtdIns(4,5)P2 very specifically (Watt et al. 2002), it is therefore unlikely that PLCη2 interacts to any great extent with this phospholipid in these cells. An interesting feature of the PtdIns(3,4,5)P3 headgroup resides in its larger size and additional negative charge compared with other phosphoinositide headgroups. This triggers its projection from the lipid bilayer which might facilitate the interaction between PtdIns(3,4,5)P3 and PH domains though little penetration of the lipid bilayer is required for docking (Chen et al. 2012). Consequently, PtdIns(3,4,5)P3-binding PLCs such as PLCη2 might represent a subgroup, which respond quicker to extracellular stimuli. Interestingly, the PLCη2 PH domain exhibited a relatively low affinity toward Ins(1,4,5)P3 as demonstrated by the high IC50 value (10.86 μM). Ins(1,4,5)P3-binding is known to regulate PLCδ1 activity, such that it competes with PtdIns(4,5)P2 to displace the PH domain (and thus the enzyme) from the plasma membrane upon its production (Hirose et al. 1999). Given the low affinity of the PLCη2 PH domain toward Ins(1,4,5)P3, it is unlikely that the hydrolyzed headgroup will be able to effectively compete with PtdIns(3,4,5)P3. Consequently, the phospholipid/phosphoinositide-binding specificity of PLCη2 likely make this enzyme particularly well-suited for the modulation of transient Ca2+-signals through sustained generation of Ins(1,4,5)P3 (and DAG) in the nucleus and in growing neurites during the differentiation process.