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Cited by 76Bone Metabolism and DiseasesCancer, Hypoxia, and MetabolismGenomics and Chromatin Dynamics

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Lactate-induced histone lactylation by p300 promotes osteoblast differentiation

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Minami, Erika, Sasa, Kiyohito, Yamada, Atsushi, Kawai, Ryota, Yoshida, Hiroshi, Nakano, Haruhisa, Maki, Koutaro, Kamijo, Ryutaro·2023·PLOS ONE
DOI 10.1371/journal.pone.0293676PMC PMC10697613
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Abstract

Lactate, which is synthesized as an end product by lactate dehydrogenase A (LDHA) from pyruvate during anaerobic glycolysis, has attracted attention for its energy metabolism and oxidant effects. A novel histone modification-mediated gene regulation mechanism termed lactylation by lactate was recently discovered. The present study examined the involvement of histone lactylation in undifferentiated cells that underwent differentiation into osteoblasts. C2C12 cells cultured in medium with a high glucose content (4500 mg/L) showed increases in marker genes (Runx2, Sp7, Tnap) indicating BMP-2-induced osteoblast differentiation and ALP staining activity, as well as histone lactylation as compared to those cultured in medium with a low glucose content (900 mg/L). Furthermore, C2C12 cells stimulated with the LDH inhibitor oxamate had reduced levels of BMP-2-induced osteoblast differentiation and histone lactylation, while addition of lactate to C2C12 cells cultured in low glucose medium resulted in partial restoration of osteoblast differentiation and histone lactylation. These results indicate that lactate synthesized by LDHA during glucose metabolism is important for osteoblast differentiation of C2C12 cells induced by BMP-2. Additionally, silencing of p300, a possible modifier of histone lactylation, also inhibited osteoblast differentiation and reduced histone lactylation. Together, these findings suggest a role of histone lactylation in promotion of undifferentiated cells to undergo differentiation into osteoblasts.

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Figures

Fig 1 High glucose concentration promoted osteoblast differentiation and increased histone lactylation levels in C2C12 cells. C2C12 cells were incubated for 72 (a right, b, c) or 24 (a left, d) hours after addition of BMP-2 (150 ng/ml) into medium with a high (4500 mg/L) or low (900 mg/L) glucose concentration. Amounts of (a) lactate, and (b) ALP staining and activity are shown. (c) Osteoblast differentiation marker genes were determined using real-time RT-PCR. (d) Expression of lactyl lysine after addition of BMP-2 was evaluated by western blotting. Data are expressed as mean ± SD (n = 3–5). *, **Significantly different from control group (*p <0.05, **p <0.01).
Fig 1 High glucose concentration promoted osteoblast differentiation and increased histone lactylation levels in C2C12 cells. C2C12 cells were incubated for 72 (a right, b, c) or 24 (a left, d) hours after addition of BMP-2 (150 ng/ml) into medium with a high (4500 mg/L) or low (900 mg/L) glucose concentration. Amounts of (a) lactate, and (b) ALP staining and activity are shown. (c) Osteoblast differentiation marker genes were determined using real-time RT-PCR. (d) Expression of lactyl lysine after addition of BMP-2 was evaluated by western blotting. Data are expressed as mean ± SD (n = 3–5). *, **Significantly different from control group (*p <0.05, **p <0.01).CC BY · PubMed Central
Fig 2 Inhibition of LDHA reduced osteoblast differentiation and lactyl lysine levels. C2C12 cells were incubated for 72 (a right, b, c) or 24 (a left, d) hours after addition of BMP-2 with oxamate. Amounts of (a) lactate, and (b) ALP staining and activity are shown. (c) Osteoblast differentiation marker genes were determined using real-time RT-PCR. (d) Expression of lactyl lysine after addition of BMP-2 and oxamate was evaluated by western blotting. Data are expressed as mean ± SD (n = 3). *, **Significantly different from control group (*p <0.05, **p <0.01). NS, not significant.
Fig 2 Inhibition of LDHA reduced osteoblast differentiation and lactyl lysine levels. C2C12 cells were incubated for 72 (a right, b, c) or 24 (a left, d) hours after addition of BMP-2 with oxamate. Amounts of (a) lactate, and (b) ALP staining and activity are shown. (c) Osteoblast differentiation marker genes were determined using real-time RT-PCR. (d) Expression of lactyl lysine after addition of BMP-2 and oxamate was evaluated by western blotting. Data are expressed as mean ± SD (n = 3). *, **Significantly different from control group (*p <0.05, **p <0.01). NS, not significant.CC BY · PubMed Central
Fig 3 Lactate recovered suppression of osteoblast differentiation and decreased histone lactylation levels caused by low glucose. C2C12 cells were incubated for 72 (a right, b, c) or 24 (a left, d) hours after placing in medium with a high or low glucose concentration in the absence or presence of lactate. Amounts of (a) lactate, and (b) ALP staining and activity are shown. (c) Osteoblast differentiation marker genes were determined using real-time RT-PCR. (d) Expression of lactyl lysine under the same conditions was evaluated by western blotting. Data are expressed as mean ± SD (n = 3–4). *, **Significantly different from control group (*p <0.05, **p <0.01). ND: not detected.
Fig 3 Lactate recovered suppression of osteoblast differentiation and decreased histone lactylation levels caused by low glucose. C2C12 cells were incubated for 72 (a right, b, c) or 24 (a left, d) hours after placing in medium with a high or low glucose concentration in the absence or presence of lactate. Amounts of (a) lactate, and (b) ALP staining and activity are shown. (c) Osteoblast differentiation marker genes were determined using real-time RT-PCR. (d) Expression of lactyl lysine under the same conditions was evaluated by western blotting. Data are expressed as mean ± SD (n = 3–4). *, **Significantly different from control group (*p <0.05, **p <0.01). ND: not detected.CC BY · PubMed Central
Fig 4 Ep300 siRNA suppressed osteoblast differentiation and decreased histone lactylation levels. Following introduction of Ep300 siRNA, C2C12 cells were cultured for 72 (a right, b, c) or 24 (a left, d) hours after addition of BMP-2 to the medium. Amounts of (a) lactate, and (b) ALP staining and activity are shown. (c) Osteoblast differentiation marker genes were determined using real-time RT-PCR. (d) Expression of lactyl lysine under the same conditions was evaluated by western blotting. Data are expressed as mean ± SD (n = 3–5). *, **Significantly different from control group (*p <0.05, **p <0.01). NS, not significant.
Fig 4 Ep300 siRNA suppressed osteoblast differentiation and decreased histone lactylation levels. Following introduction of Ep300 siRNA, C2C12 cells were cultured for 72 (a right, b, c) or 24 (a left, d) hours after addition of BMP-2 to the medium. Amounts of (a) lactate, and (b) ALP staining and activity are shown. (c) Osteoblast differentiation marker genes were determined using real-time RT-PCR. (d) Expression of lactyl lysine under the same conditions was evaluated by western blotting. Data are expressed as mean ± SD (n = 3–5). *, **Significantly different from control group (*p <0.05, **p <0.01). NS, not significant.CC BY · PubMed Central

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  1. T Katagiri, 1994, Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage, J Cell Biol, 127, 1755

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Metadata

Authors
Minami, E., Sasa, K., Yamada, A., Kawai, R., Yoshida, H., Nakano, H., Maki, K., & Kamijo, R.
Affiliations
SHOWA Medical University (JP)
Corresponding author
Sasa, Kiyohito
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2023-12-05
Volume
18
Issue
12
Pages
e0293676
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0293676
PMC
PMC10697613
OpenAlex
W4389331757
Cited by
73
References
27
Citations by year
2026: 31 · 2025: 35 · 2024: 7
FWCI
6.50
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Japan Society for the Promotion of Science (21K16936, 23K09128)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
histone lactylation, lactate, P300, osteogenic differentiation, LDHA, BMP-2, high glucose, C2C12 myoblasts, Runx2, Osterix, tissue-nonspecific alkaline phosphatase, osteoblastogenesis, histone modifications, gene expression regulation
MeSH terms
Cell Differentiation, Glucose, Histones, Osteoblasts, Lactic Acid

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