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Phospholipase C-?2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth.

ABSTRACT: Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-?2 (PLC?2) in neuritogenesis was revealed. Here we investigate the function of PLC?2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLC?2 calcium-binding EF-hand domain, a domain that is known to be required for PLC?2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLC?2 and D256A (EF-hand) and H460Q (active site) PLC?2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLC?2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLC?2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLC?2 by comparing immunoelectron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labeling for PLC?2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLC?2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of the PLC?2 EF-hand domain and articulation of PLC?2 with LIMK-1 in regulating neuritogenesis.

SUBMITTER: Arastoo M

PROVIDER: S-EPMC4735258 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth.

Arastoo Mohammed M Hacker Christian C Popovics Petra P Lucocq John M JM Stewart Alan J AJ

Histochemistry and cell biology 20151215 2

Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified n ...[more]

PMID: 26671787

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Project description:PurposeHow vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. This study sought to identify vitamin A-responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial (HCjE) cell line grown with all-trans-retinoic acid (RA). The analysis showed that secretory phospholipase A(2) group IIA (sPLA(2)-IIA) was the gene most upregulated by RA, followed by the membrane-associated mucin MUC16 at a later time point. Since eicosanoids, the product of arachidonic acid generated by the PLA(2) family, have been shown to increase mucin production, this study sought to determine whether sPLA(2) mediates the RA induction of MUC16.MethodsHCjE cells were cultured with or without RA for 3, 6, 24, and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips and analyzed using commercial software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA(2) is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad-spectrum PLA(2) inhibitor aristolochic acid (ArA) or the specific sPLA(2)-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis.ResultsAfter RA addition, 28 transcripts were upregulated and 6 downregulated by more than twofold (P < 0.01) at both 3 and 6 hours (early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours (late phase). Group IIA sPLA(2), significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA(2) upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA(2)-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced (P < 0.01).ConclusionsThe RA-associated upregulation of membrane-associated mucin MUC16 at late phase appears to be through sPLA(2)-IIA. Upregulation of this hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A facilitates maintenance of the wet-surfaced phenotype on the ocular surface.

Dataset Information

Phospholipase C-?2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth.

Publications

Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth.

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