Abstract
Neurite outgrowth, a cell differentiation process involving membrane morphological changes, is critical for neuronal network and development. The membrane lipid, phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), is a key regulator of many important cell surface events of membrane signaling, trafficking and dynamics. This lipid is produced mainly by the type I PI 4-phosphate 5-kinase (PIP5K) family members. In this study, we addressed whether PIP5Kα, an isoform of PIP5K, could have a role in neurite outgrowth induced by nerve growth factor (NGF). For this purpose, we knocked down PIP5Kα in PC12 rat pheochromocytoma cells by stable expression of PIP5Kα microRNA that significantly reduced PIP5Kα expression and PIP2 level. Interestingly, NGF-induced neurite outgrowth was more prominent in PIP5Kα-knockdown (KD) cells than in control cells. Conversely, add-back of PIP5Kα into PIP5Kα KD cells abrogated the effect of NGF on neurite outgrowth. NGF treatment activated PI 3-kinase (PI3K)/Akt pathway, which seemed to be associated with reactive oxygen species generation. Similar to the changes in neurite outgrowth, the PI3K/Akt activation by NGF was potentiated by PIP5Kα KD, but was attenuated by the reintroduction of PIP5Kα. Moreover, exogenously applied PIP2 to PIP5Kα KD cells also suppressed Akt activation by NGF. Together, our results suggest that PIP5Kα acts as a negative regulator of NGF-induced neurite outgrowth by inhibiting PI3K/Akt signaling pathway in PC12 cells.
Highlights
Neurite outgrowth is a cellular process involved in neuronal migration, differentiation and plasticity.[1]
Our results suggest that PIP5Ka acts as a negative regulator of Nerve growth factor (NGF)-induced neurite outgrowth by inhibiting PI 3-kinase (PI3K)/Akt signaling pathway in PC12 cells
PC12 cells transfected with the PIP5Ka miR- or negative control miR-expressing plasmids were selected by treatment with blasticidin
Summary
Neurite outgrowth is a cellular process involved in neuronal migration, differentiation and plasticity.[1]. The Rho family of small guanosine triphsopahatases, RhoA, Rac[1] and Cdc[42] that have critical roles in actin polymerization, function as primary regulators of neurite outgrowth.[3,4] In addition, a number of studies have demonstrated that multiple signaling events, including phosphatidylinositol (PI) 3-kinase (PI3K) and its downstream effector Akt, MAPK and reactive oxygen species (ROS) generation, participate in the mediation of neurite outgrowth.[5,6,7,8,9]. NGF is a potent inducer of neurite outgrowth.[1,10] NGF binds to the tyrosine kinase receptor TrkA, triggering activation of various signaling pathways including PI3K/Akt, phospholipase C and Ras/Raf/ MAPK cascades.[8,10,11,12,13] PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of NGF-induced neurite outgrowth. Following NGF treatment, these cells stop dividing and show terminally differentiated neuronal phenotype
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