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. 2023 Dec 5;18(12):e0293676. doi: 10.1371/journal.pone.0293676

Lactate-induced histone lactylation by p300 promotes osteoblast differentiation

Erika Minami 1,2, Kiyohito Sasa 1,*, Atsushi Yamada 1, Ryota Kawai 2, Hiroshi Yoshida 2, Haruhisa Nakano 2, Koutaro Maki 2, Ryutaro Kamijo 1
Editor: Atsushi Asakura3
PMCID: PMC10697613  PMID: 38051708

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.

Introduction

Production of lactate, a metabolite of anaerobic glycolysis, increases when demand for oxygen and ATP exceeds cellular supply, such as with exercise or infection [1]. It was previously considered to be a by-product, though is now known to be used intracellularly or released into adjacent organs as an energy source [2, 3]. Lactate has also been found to promote osteoblast differentiation via stabilization of oxia-inducible factor-1α (HIF-1α) and GPR81-PKC-Akt signaling [4, 5], while a recent study presented findings showing that lactylation by lactate is caused by a modification of histones and involved in epigenetics [6].

The term epigenetics refers to regulation of gene expression without alteration of gene sequence [7]. Typical epigenetic modifications include DNA methylation and histone modifications, with the former involving binding of methyl groups to DNA and regulation of gene expression [8–10]. On the other hand, histones are proteins that bind to DNA, and histone modifications play important roles in regulating changes in the higher-order structure of chromosomes and maintenance of cell fate through various chemical modifications, such as phosphorylation, acetylation, methylation, and ubiquitination, as well as others [11–14].

Histones are important for epigenetics, in which gene expression is regulated by acquired modifications of chromatin, and recently lactylation by lactate was found to be a novel modification of histones [6]. Lactylation is a modification in which a substructure of lactyl CoA binds to the lysine of histone proteins. As for the role of histone lactylation, when macrophages become polarized to M1 (inflammatory type) due to bacterial infection, the glycolytic system is stimulated, and lactate production and lactylation of lysine residues in histones are increased. This results in induction of gene expression necessary for polarization to M2, which is responsible for the convergence of inflammation [6]. Others have reported that the tumor metabolite lactate promotes tumorigenesis by modulating MOESIN lactation and enhancing TGF-β signaling in regulatory T cells [15] and that histone lactylation contributes to tumorigenesis by facilitating YTHDF2 expression for development of ocular melanoma [16]. In other words, lactate is not only a byproduct of glycolysis, but also an important molecule that regulates gene expression through histone modification.

In our previous study, small interfering RNA (siRNA)-mediated knockdown of monocarboxylate transporter 1 (MCT), a transporter of monocarboxylates (lactate, pyruvate, ketone bodies, etc.), was found to suppress osteoblast differentiation in the myoblast cell line C2C12 via activation of p53 [17]. It was also shown that MCT inhibition and knockdown inhibited osteoclast differentiation and function. Mct1 knockdown in macrophages promoted RANKL-induced osteoclastogenesis, whereas Mct2 knockdown inhibited it. In mature osteoclasts, Mct2 knockdown reduced the number of osteoclasts and suppressed bone resorption. Together, these results indicate that MCT1 is a negative regulator and MCT2 a positive regulator of osteoclast differentiation, as previously suggested [18]. Thus, it is considered that intracellular and extracellular transport of monocarboxylates, typified by lactate, by MCTs are key factors for differentiation of osteoblasts and osteoclasts, which are important for bone remodeling. However, the detailed mechanism of gene regulation by monocarboxylate transport for osteoblast differentiation remains largely unknown. Based on results of the present study, it is suggested that lactate production by glucose metabolism regulates osteoblast differentiation through epigenetics other than energy metabolism.

Results

High glucose level in C2C12 cells promoted osteoblast differentiation and increased histone lactylation

C2C12 cells are commonly cultured in medium with a high concentration of glucose (4500 mg/L) for maintenance and differentiation. BMP-2 stimulation inhibits C2C12 cells from differentiating into myotube cells and induces their conversion into osteoblast-like cells [19, 20]. First, to confirm changes in osteoblast differentiation due to differences in glucose metabolism, C2C12 cells were placed into high or low glucose (900 mg/L) medium, and osteoblast differentiation was induced by BMP-2. The results showed that high glucose increased both intracellular and extracellular lactate levels as compared to low glucose (Fig 1a). Furthermore, the number of alkaline phosphatase (ALP)-positive cells was increased and ALP activity enhanced when cultured in high glucose as compared to low glucose medium (Fig 1b). Interestingly, C2C12 cells cultured in high glucose medium showed upregulated expression of the transcription factors Runx2 (Runx2) and Osterix (Sp7), which are required for osteoblast differentiation, as well as of ALP (Tnap), a specific osteoblast differentiation gene (Fig 1c). Next, histone proteins from C2C12 cells cultured in high or low glucose medium were extracted, and histone lactylation was detected using an anti-lactyl-lysine antibody. The results showed that C2C12 cells cultured in high glucose medium had a greater level of histone lactylation (17 and 15 kDa bands) than those cultured in low glucose medium (Fig 1d).

Fig 1. High glucose concentration promoted osteoblast differentiation and increased histone lactylation levels in C2C12 cells.

Fig 1

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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).

Inhibition of lactate dehydrogenase A caused inhibition of osteoblast differentiation and reduced histone lactylation

Lactate dehydrogenase A (LDHA) is an enzyme that converts pyruvate produced in the glycolytic system to lactate [21], while oxamidic acid (oxamate) is an LDHA inhibitor. Oxamate was added to medium at different concentrations (0, 5, 10, 20 mM), then lactate levels in cell lysate or medium used to culture C2C12 cells were measured. Both intracellular and extracellular concentrations of lactate were found to be decreased in an oxamate concentration-dependent manner (Fig 2a). Furthermore, oxamate decreased alkaline phosphatase (ALP)-positive cells and inhibited ALP activity in a concentration-dependent manner (Fig 2b), with Sp7 and Tnap mRNA expressions in C2C12 cells were downregulated. However, Runx2 mRNA expression was not changed in C2C12 cells with addition of oxamate (Fig 2c), whereas histone lactylation levels (17 and 15 kDa bands) were reduced (Fig 2d). In contrast, histone acetylation levels were unchanged by Oxamate (S1 Fig). Histone acetylation was not detected band of 15 kDa.

Fig 2. Inhibition of LDHA reduced osteoblast differentiation and lactyl lysine levels.

Fig 2

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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.

Reduced lactate production by low glucose partially responsible for suppression of osteoblast differentiation

Next, whether lactate production is involved in osteoblast-like cell differentiation of C2C12 cells was examined by culturing in low glucose medium. First, lactate (10 or 20 mM) was added to cells cultured in low glucose medium with BMP-2, and the concentrations of intracellular and extracellular lactate in cell lysates and medium were measured(Fig 3a). Extracellular lactate in cultures of C2C12 cells cultured in low-glucose medium was increased by lactate addition, while the intracellular lactate concentration was also found to be partially recovered with addition of lactate as compared to cultures performed with high gulucose medium. Furthermore, suppression of BMP-2 introduced ALP activity in C2C12 cells induced by the low glucose medium was recovered by addition of lactate (Fig 3b). Additionally, the expression of Runx2 mRNA was completely recovered when lactate was added to cultures with low glucose as compared to high glucose medium. While the expressions of Sp7 and Tnap mRNA tended to upregulated when lactate was added as compared to low glucose medium only, those expression levels were lower than seen in high glucose medium (Fig 3c). Addition of lactate to low glucose medium also increased the histone lactylation levels (17 and 15 kDa bands) as compared to C2C12 cells cultured in low glucose medium, though those were lower than seen with high glucose medium (Fig 3d).

Fig 3. Lactate recovered suppression of osteoblast differentiation and decreased histone lactylation levels caused by low glucose.

Fig 3

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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.

Osteoblast differentiation by histone lactylation regulated by p300

Histone acetyltransferase (p300) has been reported as a possible enzyme for promoting the function of histone lactyl transferase [6, 22]. Introduction of Ep300 siRNA into C2C12 cells resulted in knockdown of Ep300 mRNA expression by approximately 75% (S2 Fig). In contrast, extracellular and intracellular lactate concentrations were not changed by Ep300 siRNA (Fig 4a). We also found that ALP-positive cells were decreased and ALP activity was suppressed with use of Ep300 siRNA (Fig 4b), while the expressions of Sp7 and Tnap mRNAs were also decreased. Although the difference was not statistically significant, expression of Runx2 mRNA was decreased in C2C12 cells by Ep300 siRNA (Fig 4c) and histone lactylation levels (17 and 15 kDa bands) were also reduced (Fig 4d). Additionally, histone acetylation levels were reduced by addition of Ep300 siRNA (S3 Fig). Histone acetylation was not detected band of 15 kDa.

Fig 4. Ep300 siRNA suppressed osteoblast differentiation and decreased histone lactylation levels.

Fig 4

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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.

Discussion

The present findings demonstrate that lactate produced by glucose metabolism promotes differentiation of cells to osteoblasts at an early stage by histone modification via p300. It is known that lactate promotes osteoblast differentiation by stabilization of hypoxia inducible factor 1a (HIF-1α) [4]. Another study that examined the pre-osteoblast cell line MC3T3-E1 found that PTH treatment-mediated osteoblast differentiation was promoted by positive feedback via GPR81-PKC-Akt signaling due to increased lactate production [5]. However, there are few reports regarding osteoblast differentiation via histone modification by lactate.

In the present study, undifferentiated C2C12 cells were used to examine changes in histone lactylation and mRNA expression during the early stage of differentiation. The results showed that increased glucose metabolism increased osteoblast differentiation and histone lactylation (Fig 1b–1d), while inhibition of LDHA decreased those (Fig 2b–2d), thus indicating that intracellular lactate production is required for osteoblast differentiation. Furthermore, osteoblast differentiation and histone lactylation, which are decreased with a low level of glucose, were partially restored by addition of lactate (Fig 3b–3d). Those results indicate that an environment that includes low glucose leads to a lower level of energy production due to reduced glycolysis, while lack of lactate production reduces osteoblast differentiation. Notably, lactate production by glucose metabolism was found to positively regulate osteoblast differentiation through p300-mediated histone lactylation (Fig 4b–4d).

Use of different glucose concentrations resulted in alterations of the mRNA expression of transcription factors such as Runx2 and Sp7, important for determining osteoblast differentiation (Fig 1c). However, oxamate and p300, which regulate histone lactylation, were not found to be involved in changes in Runx2 mRNA, though may be involved in Sp7 changes (Figs 2c and 4c). It is thus speculated that alterations in Runx2 mRNA caused by changes in glucose concentration involve actions of lactate other than lactate-induced histone lactylation, such as energy metabolism and oxidative actions. The present results indicate that histone lactylation regulates Sp7 rather than Runx2. Although Sp7 (Osterix) is a downstream gene of Runx2, a previous study found that Osterix-/- mice showed complete absence of osteoblasts, demonstrating Osterix as a transcription factor essential for osteoblast differentiation [23]. However, in the present experiments, when lactate was added to low glucose medium, Runx2 mRNA levels recovered to the same level as seen in the high glucose medium, but the increases in Sp7 and Tnap mRNA levels were small (Fig 3c). There are two possible reasons for this. First, a partial restoration of histone lactylation was noted when lactate was added to low glucose as compared to high glucose medium (Fig 3d). Second, the post-gene expression metabolism of Runx2 may be involved. A prior report noted that glucose uptake by Glut1 promotes osteoblast differentiation by suppressing AMPK-dependent proteasomal degradation of Runx2 [24] and it is conceivable that the present results may have also been due to AMPK-dependent proteasomal degradation of Runx2 by use of low glucose medium. Therefore, it is conceivable that the mRNA expression levels of Sp7 and Tnap, downstream genes of Runx2, were slightly increased by histone lactylation. Nevertheless, the detailed mechanism remains to be elucidated.

Since p300 is a histone acetyltransferase, inhibition of osteoblast differentiation by p300 knockdown may involve histone acetylation [25, 26]. Ep300 siRNA was found to decrease histone lactylation and acetylation levels (Fig 4d, S3 Fig). On the other hand, oxamate decreased only histone lactylation levels, while histone acetylation levels were not changed (Fig 2d, S1 Fig). In addition, osteoblast differentiation genes (Sp7, Tnap) were decreased in the presence of oxamate. Based on these findings, it is considered that lactate produced during the process of glucose metabolism regulates histone lactylation levels via p300 and promotes osteoblast differentiation. A recent report also noted that histone lactylation (H3K18lac) in MC3T3-E1 cells promotes osteoblast differentiation via regulation of JunB expression at the late differentiation stage [27], with the present findings providing support for those results.

In conclusion, the results of the present study show that lactate produced during glucose metabolism regulates osteoblast differentiation from undifferentiated cells through the epigenetics of histone modification.

Materials and methods

Reagents

Recombinant human BMP-2 was obtained from R&D Systems. D-glucose, L-lactic acid, and oxamate (LDHA inhibitor) were purchased from FUJIFILM Wako Pure Chemical Corporation. The PanKla antibody (PTM1401RM) and PanKac (PTM101) were obtained from PTM Biolabs and the histone H3 antibody (ab1791) from Abcam. siRNA used for knockdown of target mRNA (Silencer® Select siRNA) was obtained from Thermo Fisher Scientific, Inc.

Cell culture

C2C12 cells were purchased from RIKEN BioResource Center (C2C12 #RCB0987) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, FUJIFILM Wako Pure Chemical Corporation) supplemented with 15% fetal bovine serum (FBS) for cell proliferation. For osteoblast differentiation, C2C12 cells were stimulated with DMEM supplemented with 2.5% FBS and 150 ng/ml rhBMP-2.

ALP activity staining

C2C12 cells were seeded into 96-well plates at 1 × 104 cells/well and cultured in the presence of BMP-2 for 72 hours. Transfection of the cells with siRNA was performed at the time of seeding into the plates. Oxamate was added and pretreatment performed for two to three hours before the start of differentiation, then the culture medium was replaced with differentiation medium with Oxamate included. Cells were fixed for 30 minutes in 4% paraformaldehyde and washed with PBS, then incubated for 30 minutes at 37°C with 100 mmol/L Tris-HCl buffer (pH 8.5) containing 270 μmol/L naphthol AS-MX phosphate (Sigma-Aldrich) and 1.4 mmol/L Fast blue BB (Sigma-Aldrich). After washing with tap water, they were observed under a microscope. The scale bar shown in the figures represents 100 μm.

Determination of ALP activity

The conditions for cell culturing were the same as for ALP activity staining, noted above. For determination of ALP activity, cells were washed with PBS and homogenized with 100 μL of NP-40 under sonication. After incubation at 37°C for 30 minutes, absorbance was measured at 405 nm.

Determination of extracellular and intracellular lactate

After 24 hours or 72 hours of culturing of C2C12 cells, cell lysate or medium supernatant samples were obtained and absorbance measured at a wavelength of 450 nm using a Lactate Assay Kit-WST (Dojindo), according to the manufacturer’s instructions.

Real-time RT-PCR

Total RNA was extracted from C2C12 cells using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions, then reverse transcription reactions were performed using ReverTra ACE RT qPCR master Mix (Toyobo). Quantitative real-time RT-PCR was performed using a TaqManTM Gene Expression Assay (Gapdh, Mm99999915_g1; Tnap, Mm00475834_m1; Runx2, Mm00501584_m1; Sp7, Mm04209856_m1; Ep300, Mm00625535_m1). Gene signals were normalized against the amplified signal of Gapdh.

Western blot analysis

C2C12 cells were seeded into six-well plates at 5 × 105 cells/well and cultured in the presence of BMP-2 for 24 hours. Histone proteins were extracted from cells using an Epi Quik Total Histone Extraction Kit (Epigen Tek), according to the manufacturer’s instructions. Histone proteins were subjected to SDS-PAGE (10% polyacrylamide gel) under a reducing condition. The samples were placed in Mini-PROTEAN TGX gel and electrophoresis was performed, then the proteins were transferred to PVDF membranes and treated with primary antibodies for one hour, followed by secondary antibodies against Rabbit IgG (GE Healthcare) for on hour. Immunoreactive bands were visualized by an enhanced chemiluminescence reaction using an ECL Prime Western Blot Detection System (GE Healthcare). The intensity of the chemiluminescent bands was quantitatively analyzed using Versa Doc 5000 MP (Bio-Rad Laboratories). The ratio of the histone band intensity of lactyl lysine (Pan Kla) at approximately 17 and 15 kDa to the histone protein band intensity of total histone H3 was calculated. The band at approximately 17 kDa contained histone H2A, H2B, and H3 proteins, while that at approximately 15 kDa contained histone H4 proteins.

Statistical analysis

Values are expressed as the mean ± SD. Student’s t-test was used for comparisons between two groups. One-way ANOVA with post-hoc Tukey test was performed for comparing results from three or more groups. P-values less than 0.05 were considered to be statistically significant. All statistical analyses were performed with EZR or Microsoft® Excel® 2019 MSO. In the graph shown in Fig 3c (Sp7/Gapdh), the low glucose group was ND, thus Student’s t-test was used to compare the high glucose group with the low glucose with lactate added group.

Supporting information

S1 Fig. Inhibition of LDH was not changed histone acetylation levels in C2C12 cells.

C2C12 cells were incubated for 24 hours after addition of BMP-2 (150 ng/ml) with Oxamate. Amounts of expression of acetyl 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).

(PDF)

Click here for additional data file. (9.6KB, pdf)
S2 Fig. Ep300 siRNA decreased expression of Ep300 mRNA in C2C12 cells.

Following introduction of Ep300 siRNA, C2C12 cells were cultured for 72 hours after addition of BMP-2 to the medium. Expression of mRNA for Ep300 were analyzed by real-time PCR. Amplification signals from the gene was normalized that of Gapdh. Data are expressed as mean ± SD (n = 3). *, **Significantly different from control group (*p <0.05, **p <0.01).

(PDF)

Click here for additional data file. (10.8KB, pdf)
S3 Fig. Ep300 siRNA decreased histone acetylation levels in C2C12 cells.

Following introduction of Ep300 siRNA, C2C12 cells were cultured for 24 hours after addition of BMP-2 to the medium. Amounts of expression of acetyl lysine was evaluated by western blotting. Data are expressed as mean ± SD (n = 3). *, **Significantly different from control group (*p <0.05, **p <0.01).

(PDF)

Click here for additional data file. (29.9KB, pdf)
S1 Raw images

(PDF)

Click here for additional data file. (177.1KB, pdf)

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This work was supported by a Grant-In-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (21K16936, 23K09128). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Atsushi Asakura

20 Jul 2023

PONE-D-23-14523Lactate-induced histone lactylation by p300 promotes osteoblast differentiationPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors investigated the function and mechanism of histone lactylation on osteoblast differentiation. The results showed a role of histone lactylation in promotion of undifferentiated cells to osteoblast differentiation. The paper shows evidence that supports the conclusions, however, the following aspects should be addressed.

Major Concerns:

1. Figure1d and Figure 2d: the author only repeats once on KLA, it’s not enough in standard scientific article.

2. The author designed a LDH inhibitor (oxamate) group to study the function of LDHA on osteoblast differentiation, why not add a gene overexpression group?

3. There is no significant in results.

4. Figure 2 and 3a: Add relative intracellular lactate results.

Minor Concerns:

Figure 1b: Add scale in your picture. Check it through the MS.

Figure1d and Figure 2d: The KD of proteins should be added.

Reviewer #2: In this study, the authors demonstrated that lactate produced by glycolysis regulates osteoblast differentiation via histone H3 lactylation by promoting histone lactyl transferase activity by HAT/p300, which epigenetically enhances the expression of genes promoting osteoblast differentiation.

This is an interesting study, but some issues need to be clarified.

1. In Figures 1d through 4d, there are two bands stained with anti-lactyl lysine antibody (PanKla) in H3. What do these two bands represent and density ratio of which band was measured? In addition, there are no error bars for PanKla/H3 ratio. Because these are very important data to show H3 lactylation by lactate, these experiments need to be repeated and statistical significance should be presented.

2. In Figure 2, an LDHA inhibitor, oxamate, dose-dependently reduced extracellular lactate concentration and ALP activity, and 10 mM oxamate reduced lactyl lysine in H3 to a similar extent to that under low glucose (Figure 1d). However, Runx2 expression was inhibited only mildly and insignificantly, which is also the case in Figure 4c. Without change in Runx2 expression, how osteoblast differentiation was suppressed by oxamate?

3. In Figure 3, addition of lactate to low glucose medium recovered extracellular lactate concentration, ALP activity, PanKla/H3 ratio and Runx2 expression. In contrast, there was almost no change in Sp7 and Tnap expression. How ALP activity was increased despite very little change in Tnap expression, and what was the explanation for these discrepant results in Runx2, Sp7 and Tnap between Figures2 and 3? In Figures 2a and 3a, only extracellular lactate concentration was demonstrated. However, what is important is intracellular lactate concentration, and the authors should demonstrate intracellular lactate concentration.

4. In Figure 4, gene expression profiles of Runx2, Sp7 and Tnap were similar to those under oxamate treatment in Figure 2, and were different from those in Figure 3. These discrepant results under different treatments give readers a concern about the validity of the authors’ conclusion. In addition, knockdown of p300/HAT causes inhibition of histone acetylation and may change expression of various genes aside from change in histone lactylation.

5. It is worthwhile to refer to and discuss about the paper by Karsenty group (Wei J, et al. Cell 151: 1576-91, 2015) demonstrating that glucose uptake via Runx2-dependent expression of Glut1 is required and that Glut1 and Runx2 crosstalk determines osteoblast differentiation.

Reviewer #3: The study by Minami et al. describes the role of p300 lactylation as a mechanism associated with osteoblast differentiation. The authors conducted an experimental design using C2C12 cells to demonstrate the involvement of lactate in BMP-2-induced differentiation. While the experiments support this mechanism, there are some aspects that should be evaluated by the authors.

Considering that histone lactylation is key to understanding the regulatory mechanism in this article, it is noteworthy that only one result is shown in the bar graph of Figures 1, 2, 3, and 4 (d). It is suggested to include more replicates and display their dispersion as standard deviation (SD) along with statistical analysis. Were both bands considered for relative quantification since two bands are observed? Whatever the case may be, it is recommended to clarify this in the Materials and Methods section.

To demonstrate the role of LDHA and the conversion of pyruvate to lactate, oxamate was used. Was a cell viability analysis performed to rule out possible cytotoxic effects?

For the statistical analyses, the Student's t-test was used. However, in Figures 2 and 3, more than two experimental groups were used. In such cases, it is more appropriate to use analysis of variance (ANOVA) and post-hoc multiple comparisons tests. Additionally, significant differences are indicated in Figure 3C (Tnap/Gapdh) between low/0 and low/20 mM lactate, but the graph should be modified on its axis to visualize this unclear difference. Furthermore, in this figure, differences between high and low are shown for the Sp7/Gapdh gene, but no expression was detected in one group. It should be clearly stated which groups are being compared in this figure.

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Reviewer #2: No

Reviewer #3: No

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PLoS One. 2023 Dec 5;18(12):e0293676. doi: 10.1371/journal.pone.0293676.r002

Author response to Decision Letter 0

27 Aug 2023

Reply to Reviewers

Revision Notes

We sincerely appreciate the helpful comments from the reviewers in regard to our study, which provided new insights. As a result, additional experiments were performed according to the editor’s and reviewers’ suggestions, and revisions have been made, resulting in great improvements to the manuscript.

Reviewer 1

The reviewer’s comments and suggestions are sincerely appreciated, and were helpful to improve our study. Please note our responses following.

Major Concerns:

Comment #1:

Figure1d and Figure 2d: the author only repeats once on KLA, it’s not enough in standard scientific article.

Our reply to comment #1

This suggestion is greatly appreciated. We carefully reperformed the experiments (n=3), and added the findings as Figures 1d, 2d, 3d, and 4d in the revised version.

Comment #2:

The author designed a LDH inhibitor (oxamate) group to study the function of LDHA on osteoblast differentiation, why not add a gene overexpression group?

Our reply to comment #2

Thank you for pointing this out. The experiments were performed by introducing plasmid DNA into C2C12 cells. The results showed that overexpression of LDHA in C2C12 cells did not promote osteoblastic differentiation. Previous studies have shown that LdhA is highly expressed in muscle tissues [Mishra et al., Cancers (Basel). 11(6): 750, 2019., Markert, et al., Science 11;189(4197):102-14, 1975.]. Since C2C12 cells are mouse myoblasts, it is possible that overexpression of LdhA had little effect on osteoblast differentiation due to the high expression level of LdhA mRNA (Appendix Fig. 1).

Comment #3:

There is no significant in results.

Our reply to comment #3

The authors are not sure if the reviewer is referring to the results shown in Figure 2c (Runx2/Gapdh) or those in Figure 4a, c (Runx2/Gapdh). With confirmation, we will do our best to respond to the comment.

Comment #4:

Figure 2 and 3a: Add relative intracellular lactate results.

Our reply to comment #4

We appreciate this helpful suggestion. Intracellular lactate graphs have been added to the revised version of the manuscript (Fig. 2a, 3a). The intracellular lactate concentration was found to be correlated with histone lactylation.

Minor Concerns:

Comment #1:

Figure 1b: Add scale in your picture. Check it through the MS.

Our reply to comment #1

A scale bar indicating 100 μm has been added the photographs have also been replaced (Fig. 1b, 2b, 3b, 4b). Related details are now described in the Materials and Methods section of the revised version (page 12, lines 278-279).

Comment #2:

Figure1d and Figure 2d: The KD of proteins should be added.

Our reply to comment #2

Protein kDa values have been added to the photographs (Fig. 1d, 2d, 3d, 4d). Related details are now described in the Materials and Methods section of the revised version (page 14, lines 311-315).

Reviewer 2

We greatly appreciate the reviewer’s comments and suggestions regarding our study. Please note our responses following. We consider that the manuscript has been significantly improved.

Comment #1:

In Figures 1d through 4d, there are two bands stained with anti-lactyl lysine antibody (PanKla) in H3. What do these two bands represent and density ratio of which band was measured? In addition, there are no error bars for PanKla/H3 ratio. Because these are very important data to show H3 lactylation by lactate, these experiments need to be repeated and statistical significance should be presented.

Our reply to comment #1

Thank you for pointing this out. The density ratio of the upper band is approximately 17 kDa and it contains histones H2A, H2B, and H3, while that of the lower band is approximately 15kDa and it contains H4. As suggested by the reviewer, additional experiments (n=3) were conducted, with the findings shown in a graph after separating the approximately 17 and 15 kDa bands. These results are described in the Materials and Methods section of the revised manuscript (page 14, lines 311-315).

Comment #2:

In Figure 2, an LDHA inhibitor, oxamate, dose-dependently reduced extracellular lactate concentration and ALP activity, and 10 mM oxamate reduced lactyl lysine in H3 to a similar extent to that under low glucose (Figure 1d). However, Runx2 expression was inhibited only mildly and insignificantly, which is also the case in Figure 4c. Without change in Runx2 expression, how osteoblast differentiation was suppressed by oxamate?

Our reply to comment #2

The authors appreciate the comments from the reviewer and this helpful question. There are two important transcription factors related to differentiation of mesenchymal stem cells into osteoblasts, Runx2 and Osterix (Sp7). Osterix is a downstream gene of Runx2, though a previous study found that Osterix-/- mice showed complete absence of osteoblasts, demonstrating that Osterix is a transcription factor essential for osteoblast differentiation [Nakashima, et al., Cell. 11;108(1):17-29, 2002.]. Based on these findings, we consider that a reduction of intracellular lactate by oxamate suppresses osteoblast differentiation through reduction of Osterix gene expression. Comments in this regard have been added to the revised Discussion section (page 10, lines 221-225).

Comment #3:

1) In Figure 3, addition of lactate to low glucose medium recovered extracellular lactate concentration, ALP activity, PanKla/H3 ratio and Runx2 expression. In contrast, there was almost no change in Sp7 and Tnap expression.

2) How ALP activity was increased despite very little change in Tnap expression, and what was the explanation for these discrepant results in Runx2, Sp7 and Tnap between Figures2 and 3?

3) In Figures 2a and 3a, only extracellular lactate concentration was demonstrated. However, what is important is intracellular lactate concentration, and the authors should demonstrate intracellular lactate concentration.

Our reply to comment #3

Thank you for these important suggestions and questions.

3-1) As noted regarding the difference between ALP activity and Tnap gene expression, the data shown in the Tnap/Gapdh and ALP activity graphs do not correlate with those obtained on 72 hours of culture. We newly checked gene expression at 24 hours after differentiation (Appendix Fig. 2), which adding lactate to low glucose medium showed that the Sp7 and Tnap mRNA levels were nearly the same as those in high glucose medium, while the mRNA levels of Runx2 were the same in nearly all of the groups. It is possible that the ALP activity graph may reflect gene expression in the early phase of culturing, though we do not have details in that regard to show.

3-2) We concluded that histone lactylation regulates Sp7 (Osterix) rather than Runx2, because neither oxamate stimulation nor Ep300 siRNA altered the Runx2 gene (Fig. 2c, 4c). Thus, it is considered that factors other than histone lactylation are involved in the increase in the Runx2 gene seen with the addition of lactate to low glucose medium. For example, the presence of a Gαi protein coupled receptor (GPR81), a lactate receptor, present on the cell membrane. A previous report noted that lactate increased Runx2 gene expression via GPR81-Akt signaling in MC3T3-E1 cells [Yu Wu, et al., Biochem Biophys Res Commun. 5;503(2):737-743, 2018.]. However, in the present experiments, addition of lactate to low glucose medium resulted in a slight increase in expression levels of Sp7 and Tnap (Fig. 3c). There are two possible reasons for this. First, a partial restoration of histone lactylation was noted when lactate was added to the low glucose as compared to the high glucose medium (Fig. 3d). Second, the post-gene expression metabolism of Runx2 may be involved. These factors are presented in the paper noted in Comment 5 from the reviewer below. Those authors noted that glucose uptake by Glut1 promotes osteoblast differentiation by suppressing the AMPK-dependent proteasomal degradation of Runx2 [Wei J, et al., Cell 151: 1576-91, 2015]. We found that the addition of lactate to low glucose medium restored Runx2 gene expression to a level similar to that noted with the high glucose medium. However, complete recovery of Sp7 and Tnap mRNA levels was not observed, which may have been caused by promotion of the AMPK-dependent proteasomal degradation of Runx2 due to decreased glucose uptake in low glucose medium. Therefore, it is conceivable that the mRNA expression levels of Sp7 and Tnap, downstream genes of Runx2, were slightly increased by histone lactylation. However, the detailed mechanism is not well understood. These issues are now discussed in the Discussion section of the revised manuscript (page 10-11, lines 225-237).

3-3) This suggestion is greatly appreciated. We have added intracellular lactate graphs (Fig. 2a, 3a), which show a correlation of intracellular lactate concentration with histone lactylation.

Comment #4:

1) In Figure 4, gene expression profiles of Runx2, Sp7 and Tnap were similar to those under oxamate treatment in Figure 2 and were different from those in Figure 3. These discrepant results under different treatments give readers a concern about the validity of the authors’ conclusion.

2) In addition, knockdown of p300/HAT causes inhibition of histone acetylation and may change expression of various genes aside from change in histone lactylation.

Our reply to comment #4

4-1) As described in our reply to Comment 3, we consider that the change in Runx2 gene expression caused by addition of lactate to low glucose medium was not caused by an increase in histone lactylation, but rather by other factors.

4-2) As noted by the reviewer, Ep300 siRNA must also be considered to regulate gene expression by histone acetylation. Therefore, we conducted additional experiments with various amounts of protein for PanKac. Ep300 siRNA decreased both histone lactylation and histone acetylation, whereas addition of oxamate did not cause a change in histone acetylation (Fig. 4d, Fig. S1, Fig. S3). On the other hand, oxamate suppressed osteoblast differentiation (Fig. 2b,c). Based on these findings, lactate produced by glucose metabolism may promote osteoblast differentiation through histone lactylation rather than histone acetylation via p300. These findings are presented in the revised Discussion section (page 11, lines 239-245).

Comment #5:

It is worthwhile to refer to and discuss about the paper by Karsenty group (Wei J, et al. Cell 151: 1576-91, 2015) demonstrating that glucose uptake via Runx2-dependent expression of Glut1 is required and that Glut1 and Runx2 crosstalk determines osteoblast differentiation.

Our reply to comment #5

Thank you for this good suggestion. Please see our reply to comment #3-2.

Reviewer 3

The comments regarding our manuscript are greatly appreciated and were very helpful. Please note our responses to the suggestions following.

Comment #1:

Considering that histone lactylation is key to understanding the regulatory mechanism in this article, it is noteworthy that only one result is shown in the bar graph of Figures 1, 2, 3, and 4 (d). It is suggested to include more replicates and display their dispersion as standard deviation (SD) along with statistical analysis. Were both bands considered for relative quantification since two bands are observed? Whatever the case may be, it is recommended to clarify this in the Materials and Methods section.

Our reply to comment #1

Thank you for the good question pointing out these issues. The upper band is approximately 17 kDa and contains histones H2A, H2B and H3, while the lower band is approximately 15kDa and contains histone H4. Based on the reviewer’s comments, we conducted additional experiments (n=3) and created a graph by separating the approximately 17 and 15 kDa bands. Those details are presented in the revised Materials and Methods section (page 14, lines 311-315).

Comment #2:

To demonstrate the role of LDHA and the conversion of pyruvate to lactate, oxamate was used. Was a cell viability analysis performed to rule out possible cytotoxic effects?

Our reply to comment #2

Thank you for this good question. To investigate the cytotoxicity of oxamate, an MTS assay was performed on 72 hours of culture, though no significant change was observed. Those results showed that oxamate (10 mM) had low toxicity (Appendix Fig. 3).

Comment #3:

For the statistical analyses, the Student's t-test was used. However, in Figures 2 and 3, more than two experimental groups were used. In such cases, it is more appropriate to use analysis of variance (ANOVA) and post-hoc multiple comparisons tests.

Our reply to comment #3

Thank you for these helpful comments. According, we performed one-way ANOVA with a post-hoc Tukey test to compare results from more than two groups. A related description has been added to the Statistical analysis section of the revised manuscript (page 14, lines 318-322).

Comment #4:

Additionally, significant differences are indicated in Figure 3C (Tnap/Gapdh) between low/0 and low/20 mM lactate, but the graph should be modified on its axis to visualize this unclear difference. Furthermore, in this figure, differences between high and low are shown for the Sp7/Gapdh gene, but no expression was detected in one group. It should be clearly stated which groups are being compared in this figure.

Our reply to comment #4

Changed the vertical axis of the graph to logarithmic scale (Fig3c:Tnap/Gapdh).

Based on these helpful suggestions, the graph in Figure 3c showing Sp7/Gapdh was examined again. The low glucose group was ND, so Student’s t-test was used to compare the high and the low glucose medium after adding lactate. A related description has been added to the revised Statistical analysis section (page 14, lines 322-324). In addition, also in Figure 3c (Tnap/Gapdh), the significant difference has been revised because we changed from Student’s t-test to Tukey test results (low glucose medium vs low glucose medium with added lactate).

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Decision Letter 1

Atsushi Asakura

18 Oct 2023

Lactate-induced histone lactylation by p300 promotes osteoblast differentiation

PONE-D-23-14523R1

Dear Dr. Sasa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Atsushi Asakura, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: My comments were adequately addressed by the authors. The quality of the manuscript was improved after revision.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Reviewer #1: Yes: Xiangzhen Shen

Reviewer #2: No

Reviewer #3: No

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Acceptance letter

Atsushi Asakura

26 Oct 2023

PONE-D-23-14523R1

Lactate-induced histone lactylation by p300 promotes osteoblast differentiation

Dear Dr. Sasa:

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on behalf of

Dr. Atsushi Asakura

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Inhibition of LDH was not changed histone acetylation levels in C2C12 cells.

    C2C12 cells were incubated for 24 hours after addition of BMP-2 (150 ng/ml) with Oxamate. Amounts of expression of acetyl 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).

    (PDF)

    Click here for additional data file. (9.6KB, pdf)
    S2 Fig. Ep300 siRNA decreased expression of Ep300 mRNA in C2C12 cells.

    Following introduction of Ep300 siRNA, C2C12 cells were cultured for 72 hours after addition of BMP-2 to the medium. Expression of mRNA for Ep300 were analyzed by real-time PCR. Amplification signals from the gene was normalized that of Gapdh. Data are expressed as mean ± SD (n = 3). *, **Significantly different from control group (*p <0.05, **p <0.01).

    (PDF)

    Click here for additional data file. (10.8KB, pdf)
    S3 Fig. Ep300 siRNA decreased histone acetylation levels in C2C12 cells.

    Following introduction of Ep300 siRNA, C2C12 cells were cultured for 24 hours after addition of BMP-2 to the medium. Amounts of expression of acetyl lysine was evaluated by western blotting. Data are expressed as mean ± SD (n = 3). *, **Significantly different from control group (*p <0.05, **p <0.01).

    (PDF)

    Click here for additional data file. (29.9KB, pdf)
    S1 Raw images

    (PDF)

    Click here for additional data file. (177.1KB, pdf)
    Attachment

    Submitted filename: Reply to Reviewers.docx

    Click here for additional data file. (32.2KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting information files.

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