Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-αl variants in embryonic mouse brain

FASEB J. 2019 Jan;33(1):808-820. doi: 10.1096/fj.201800359R. Epub 2018 Jul 31.

Abstract

Carboxypeptidase E (CPE), an exopeptidase involved in proneuropeptide processing, is also a neurotrophic factor, named neurotrophic factor-α1 (NF-α1) and has important roles in neuroprotection, stem cell differentiation, and neurite outgrowth, independent of enzymatic activity. Additionally, an N-terminal-truncated CPE/NF-α1 variant, (CPE/NF-α1)-ΔN, proposed from bioinformatic analysis of GenBank (National Center for Biotechnology Information, Bethesda, MD, USA) DNA sequences and encoding a 40-kDa protein, has been found to be exclusively expressed in embryonic neurons. To investigate the function of (CPE/NF-α1)-ΔN in neurodevelopment, we first cloned (CPE/NF-α1)-ΔN transcripts from an embryonic mouse brain. A rapid amplification of cDNA ends assay, DNA sequencing, and Northern blot revealed 1.9- and 1.73-kb transcripts, which encoded 47- and 40-kDa (CPE/NF-α1)-ΔN proteins, respectively. Those proteins were expressed in embryonic mouse brain. Expression of the 2 (CPE/NF-α1)-ΔN mRNAs surged at embryonic d 10.5, correlating with the time of neurogenesis in the developing brain and also at postnatal d 1. HT22 cells, a mouse hippocampal cell line, transduced with 40 kDa (CPE/NF-α1)-ΔN up-regulated expression of genes involved in embryonic neurodevelopment: insulin-like growth factor binding protein 2 ( IGFBP2), death-associated protein 1, and ephrin A1, which regulate proliferation, programmed cell death, and neuronal migration, respectively. HT22 cells and embryonic cortical neurons overexpressing 40 kDa (CPE/NF-α1)-ΔN exhibited enhanced proliferation, which was inhibited by IGFBP2 short interfering RNA treatment. Thus, 40 kDa (CPE/NF-α1)-ΔN has an important, enzymatically independent role in the regulation of genes critical for neurodevelopment.-Xiao, L., Yang, X., Sharma, V. K., Loh, Y. P. Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-αl variants in embryonic mouse brain.

Keywords: death associated protein-1; ephrin A1; insulin-like growth factor binding protein 2; neurodevelopment.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blotting, Northern
  • Brain / cytology
  • Brain / embryology*
  • Brain / metabolism
  • Carboxypeptidase H / genetics
  • Carboxypeptidase H / metabolism*
  • Cell Line
  • Cell Proliferation*
  • Cloning, Molecular*
  • Gene Expression Regulation, Developmental*
  • Hydrogen-Ion Concentration
  • Insulin-Like Growth Factor Binding Protein 2 / genetics
  • Insulin-Like Growth Factor Binding Protein 2 / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Nerve Growth Factors / metabolism*
  • Neurons / cytology*
  • Oligonucleotide Array Sequence Analysis
  • RNA, Messenger / genetics
  • Up-Regulation

Substances

  • Insulin-Like Growth Factor Binding Protein 2
  • Nerve Growth Factors
  • RNA, Messenger
  • Carboxypeptidase H