TY - JOUR
T1 - ZSWIM8 is a myogenic protein that partly prevents C2C12 differentiation
AU - Okumura, Fumihiko
AU - Oki, Nodoka
AU - Fujiki, Yuha
AU - Ikuta, Rio
AU - Osaki, Kana
AU - Hamada, Shun
AU - Nakatsukasa, Kunio
AU - Hisamoto, Naoki
AU - Hara, Taichi
AU - Kamura, Takumi
N1 - Funding Information:
MCF10A cells were a gift from Dr. Chin Ha Chung (Seoul National University, Korea). C2C12 cells were a gift from Dr. Masafumi Kuzuya (Nagoya University, Japan). This work was supported in parts by JSPS KAKENHI Grant Numbers 17H03652 (to F.O., K.N., and T.K.) and 18K06213 (to F.O.), the Takeda Science Foundation (to F.O.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Uehara Memorial Foundation (to F.O.), the Inamori Foundation (to F.O.), the Ichiro Kanehara Foundation (to F.O.), the Sumitomo Foundation (to F.O.), and Fukuoka Women’s University (to F.O.).
Funding Information:
Cell culture and transduction. HEK293T, 786-O, K562, RCC4, and U-2 OS cells (American Type Culture Collection, ATCC) were cultured in DMEM (Fujifilm Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FBS), penicillin G (100 units/mL), and streptomycin (100 μg/mL). C2C12 cells were a gift from Dr. Masafumi Kuzuya (Nagoya University, Japan). C2C12 cells (RCB0987, lot #41) were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. MCF10A cells were cultured in DMEM/Ham’s F-12 (Fujifilm Wako Pure Chemical Corporation) supplemented with 5% FBS, 100 units/ mL penicillin G (Nakalai, Japan), 100 μg/mL streptomycin (Nakalai, Japan), 20 ng/mL epidermal growth factor (Novus Biologicals, USA), 10 μg/mL insulin (Roche, Germany), 1 ng/mL cholera toxin (Bio Academia, Japan), and 1 μg/mL hydrocortisone (Tokyo Chemical Industry). Confluent C2C12 cells on gelatin-coated dishes were differentiated into myotubes by replacing 10% fetal calf serum (FCS) with 2% horse serum. The medium was changed every day for the indicated periods. MG132 (2 μM) was added to the culture medium for the indicated period. HEK293T and C2C12 cells were transfected with expression plasmids using polyethyleneimine (MW-25 K; Polysciences Inc., USA) at a mass ratio of 1:3.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - Cell adhesion molecule-related/downregulated by oncogenes (Cdon) is a cell-surface receptor that mediates cell–cell interactions and positively regulates myogenesis. The cytoplasmic region of Cdon interacts with other proteins to form a Cdon/JLP/Bnip-2/CDC42 complex that activates p38 mitogen-activated protein kinase (MAPK) and induces myogenesis. However, Cdon complex may include other proteins during myogenesis. In this study, we found that Cullin 2-interacting protein zinc finger SWIM type containing 8 (ZSWIM8) ubiquitin ligase is induced during C2C12 differentiation and is included in the Cdon complex. We knocked-down Zswim8 in C2C12 cells to determine the effect of ZSWIM8 on differentiation. However, we detected neither ZSWIM8-dependent ubiquitination nor the degradation of Bnip2, Cdon, or JLP. In contrast, ZSWIM8 knockdown accelerated C2C12 differentiation. These results suggest that ZSWIM8 is a Cdon complex-included myogenic protein that prevents C2C12 differentiation without affecting the stability of Bnip2, Cdon, and JLP.
AB - Cell adhesion molecule-related/downregulated by oncogenes (Cdon) is a cell-surface receptor that mediates cell–cell interactions and positively regulates myogenesis. The cytoplasmic region of Cdon interacts with other proteins to form a Cdon/JLP/Bnip-2/CDC42 complex that activates p38 mitogen-activated protein kinase (MAPK) and induces myogenesis. However, Cdon complex may include other proteins during myogenesis. In this study, we found that Cullin 2-interacting protein zinc finger SWIM type containing 8 (ZSWIM8) ubiquitin ligase is induced during C2C12 differentiation and is included in the Cdon complex. We knocked-down Zswim8 in C2C12 cells to determine the effect of ZSWIM8 on differentiation. However, we detected neither ZSWIM8-dependent ubiquitination nor the degradation of Bnip2, Cdon, or JLP. In contrast, ZSWIM8 knockdown accelerated C2C12 differentiation. These results suggest that ZSWIM8 is a Cdon complex-included myogenic protein that prevents C2C12 differentiation without affecting the stability of Bnip2, Cdon, and JLP.
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U2 - 10.1038/s41598-021-00306-6
DO - 10.1038/s41598-021-00306-6
M3 - Article
C2 - 34686700
AN - SCOPUS:85117719885
SN - 2045-2322
VL - 11
JO - Scientific reports
JF - Scientific reports
IS - 1
M1 - 20880
ER -