Abstract
In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production. The neuronal differentiation in nip1-overexpressing P19 cells was achieved in a retinoic acid-independent manner and was corroborated by an increase in the expression of the neuronal basic helix-loop-helix transcription factors and neural-lineage cell markers. Furthermore, depletion of nip1 by short hairpin RNA led to a decrease in the expression of neuronal basic helix-loop-helix transcription factors and ROS. However, inhibition of ROS production in nip1-overexpressing P19 cells restricted but did not extinguish neuronal differentiation. Microarray and mass spectrometry analysis identified intermediate filaments as the principal cytoskeletal elements affected by up-regulation of nip1. We show here the first evidence for a functional interaction between Nip1 and a component of the nuclear lamina, lamin A/C. associated with a neuronal-specific phenotype. Taken together, our data reveal an important role for Nip1 in the guidance of neuronal differentiation through ROS generation and modulation of intermediate filaments and implicate Nip1 as a novel intrinsic regulator of neuronal cell fate.
Highlights
Embryonic neural stem (NS)7 cells and adult neural stem cells reside in specific tissue niches such as hippocampal subgranular and subventricular zones
We show that nip1 is expressed in brain tissue
Because Numb-interacting protein 1 (Nip1) was up-regulated in primary NS cells and developing brain tissue, we investigated the role of Nip1 in regulating mammalian stem cell differentiation toward the neuronal lineages
Summary
Plasmids and Expression Constructs—A 1.6-kb cDNA fragment containing the complete coding sequence of mouse nip (BC019755) tagged with a c-Myc epitope was inserted into the phosphoglycerate kinase (PGK) vector via the SmaI/Xho restriction sites. The nip short hairpin RNA (shRNA) expression constructs consisted of pairs of short hairpin DNA sequences complementary to regions of the nip gene (see supplemental Table 1) inserted into the mU6Pro vector (a gift from Dave Turner, University of Michigan, Ann Arbor, MI) after digestion with Bbs/1 and Xho as previously described [28]. A scrambled sequence (previously described in Ref. 29) was inserted into the mU6Pro vector to serve as a negative control. For suppression of Nip expression, P19 cells were transfected, using FuGENE 6, with the mU6pro vector containing shRNA oligos directed against two different regions of the nip gene. Cells were isolated and induced to differentiate into neurons marked by III tubulin expression (supplemental Fig. S2B) on polyornithine-coated dishes as previously described [32]. Statistical Analysis—Statistical analysis was performed with Student’s t test. p values of p Ͻ 0.05 are indicated as asterisks and are considered statistically significant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
