Research Paper Volume 14, Issue 7 pp 2966—2988

Downregulation of IGFBP5 contributes to replicative senescence via ERK2 activation in mouse embryonic fibroblasts

Iyori Nojima1, , Ryusuke Hosoda1, , Yuki Toda1, , Yoshiki Saito1, , Naohiro Ueda1, , Kouhei Horimoto1, , Naotoshi Iwahara1, , Yoshiyuki Horio1, , Atsushi Kuno1, ,

  • 1 Department of Pharmacology, Sapporo Medical University School of Medicine, Sapporo, Japan

Received: July 1, 2021       Accepted: March 23, 2022       Published: April 4, 2022
How to Cite

Copyright: © 2022 Nojima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Insulin-like growth factor (IGF)-binding proteins (IGFBPs) are secretory proteins that regulate IGF signaling. In this study, we investigated the role of IGFBP5 in replicative senescence in embryonic mouse fibroblasts (MEFs). During passages according to the 3T3 method, MEFs underwent senescence after the 5th passage (P5) based on cell growth arrest, an increase in the number of cells positive for senescence-associated β-galactosidase (SA-β-GAL) staining, and upregulation of p16 and p19. In P8 MEFs, IGFBP5 mRNA level was markedly reduced compared with that in P2 MEFs. Downregulation of IGFBP5 via siRNA in P2 MEFs increased the number of SA-β-GAL-positive cells, upregulated p16 and p19, and inhibited cell growth. Incubation of MEFs with IGFBP5 during serial passage increased the cumulative population doubling and decreased SA-β-GAL positivity compared with those in vehicle-treated cells. IGFBP5 knockdown in P2 MEFs increased phosphorylation levels of ERK1 and ERK2. Silencing of ERK2, but not that of ERK1, blocked the increase in the number of SA-β-GAL-positive cells in IGFBP5-knockdown cells. The reduction in the cell number and upregulation of p16 and p21 in IGFBP5-knockdown cells were attenuated by ERK2 knockdown. Our results suggest that downregulation of IGFBP5 during serial passage contributes to replicative senescence via ERK2 in MEFs.


CDK: cyclin-dependent kinase; ERK: extracellular signal-regulated kinase; MEF: mouse embryonic fibroblast; GSK3β: glycogen synthase kinase 3β; H3K27: histone H3 lysine 27; HUVEC: human umbilical endothelial cell; IGF-1: insulin/insulin-like growth factor-1; IGFBP: IGF-binding protein; PRC1/2: polycomb repressor complexes 1/2; RT-qPCR: reverse transcription-quantitative polymerase chain reaction; SA-β-GAL: senescence-associated β-galactosidase; siRNA: small interfering RNA; TFEB: transcription factor EB; TGFβ: transforming growth factor β.