Skeletal progenitors preserve proliferation and self-renewal upon inhibition of mitochondrial respiration by rerouting the TCA cycle

Abstract

A functional electron transport chain (ETC) is crucial for supporting bioenergetics and biosynthesis. Accordingly, ETC inhibition decreases proliferation in cancer cells but does not seem to impair stem cell proliferation. However, it remains unclear how stem cells metabolically adapt. In this study, we show that pharmacological inhibition of complex III of the ETC in skeletal stem and progenitor cells induces glycolysis side pathways and reroutes the tricarboxylic acid (TCA) cycle to regenerate NAD⁺ and preserve cell proliferation. These metabolic changes also culminate in increased succinate and 2-hydroxyglutarate levels that inhibit Ten-eleven translocation (TET) DNA demethylase activity, thereby preserving self-renewal and multilineage potential. Mechanistically, mitochondrial malate dehydrogenase and reverse succinate dehydrogenase activity proved to be essential for the metabolic rewiring in response to ETC inhibition. Together, these data show that the metabolic plasticity of skeletal stem and progenitor cells allows them to bypass ETC blockade and preserve their self-renewal.

Keywords: CP: Metabolism; CP: Stem cell research; NAD regeneration; TCA rerouting; TET activity; cell-based regenerative medicine; electron transport chain; metabolic plasticity; proliferation; reverse succinate dehydrogenase; self-renewal; skeletal stem cells.

Graphical abstract

Figure 1

SSPCs preserve proliferation, self-renewal, and trilineage potential upon ETC dysfunction (A) Flow-cytometry analysis of BrdU-positive (BrdU⁺) mouse periosteal cells (mPDCs) treated for 24 h or 3 weeks with vehicle (veh) or 10 μM antimycin A (AMA) (n = 3). (B) Cell viability of veh- and AMA-treated mPDCs, analyzed by annexin V-propidium iodide flow cytometry (n = 5). (C and D) Visualization (methylene blue staining) and quantification of primary colonies, formed by mPDCs treated with veh or AMA for 1 week (n = 8). (E) Flow-cytometry analysis of the percentage of skeletal stem cells (SSCs) in freshly isolated mPDCs (fresh) or after treatment with veh or AMA for 1 week (n = 6). (F) Flow-cytometry analysis of the percentage of pre-bone, cartilage, and stromal progenitors (pre-BCSP) in freshly isolated mPDCs or after treatment with veh or AMA for 1 week (n = 6). (G–J) Visualization (methylene blue staining) and quantification of primary (G and H; n = 10) and secondary (I and J; n = 4) colonies, formed by mPDCs treated with veh or AMA for 3 weeks. (K and L) Flow-cytometry analysis of the percentage of SSCs (K) and pre-BCSPs (L) in mPDCs treated with veh or AMA for 3 weeks (n = 8). (M and N) Chondrogenic differentiation of mPDCs treated with veh or AMA for 3 weeks and assessed by visualization and quantification of chondrogenic matrix (Alcian blue staining; M, n = 6) and Col2a1 and Acan mRNA levels (N, n = 5). (O and P) Osteogenic differentiation of mPDCs treated with veh or AMA for 3 weeks and assessed by staining for alkaline phosphatase activity (ALP) and mineralized matrix (Alizarin red staining; O, n = 5) and quantification of Runx2, Tnap, and Col1a1 mRNA levels (P, n = 5–6). (Q and R) Adipogenic differentiation of mPDCs treated with veh or AMA for 3 weeks and assessed by visualization of lipid deposits (oil red O staining; Q, n = 10) and quantification of Pparγ mRNA levels (R, n = 6). Data are means ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 versus vehicle (paired two-tailed Student’s t test). Scale bars, 1 cm (C, G, I, M, O) and 200 μm (Q). See also Figures S1 and S2.

Figure 2

SSPCs increase NAD(H) pool and NAD⁺ regeneration by glycolysis side pathway and TCA cycle rerouting (A) Flow-cytometry analysis of BrdU⁺ SSPCs treated with vehicle (veh) or 10 μM AMA for 12 h or 24 h (n = 3). (B) NAD⁺ levels after treatment for indicated times (n = 4). (C) Nampt mRNA levels after treatment for indicated times (n = 4). (D) Flow-cytometry analysis of BrdU⁺ SSPCs treated for 24 h or 48 h with veh or AMA with or without 10 nM FK866 (n = 8). (E) Metabolite levels of pyruvate, lactate, alanine, and glycerol-3-phosphate (glycerol-3-P) after treatment for 3 days (n = 8). (F) Glycolytic flux (n = 3). (G) Fractional contribution of ¹³C₆-glucose to lactate, alanine, and glycerol-3-P (n = 8). (H) Metabolite levels of citrate, α-ketoglutarate (αKG), succinate (suc), fumarate (fum), malate, and 2-hydroxyglutarate (2HG) after treatment for 3 days (n = 11–15). (I) Fractional contribution of ¹³C₅-glutamine to citrate, αKG, suc, fum, malate, and 2HG (n = 8). (J) Schematic representation of ¹³C₅-glutamine labeling patterns. Empty circles indicate ¹²C, black circles denote ¹³C derived from oxidative metabolism, and gray circles denote ¹³C derived from reductive metabolism. (K and L) Oxidative (K) and reductive (L) contribution of ¹³C₅-glutamine to suc, fum, malate, and citrate (n = 8). (M) Fractional contribution of ¹³C₆-glucose to citrate, αKG, suc, fum, malate, and 2HG (n = 8). (N) Schematic representation of ¹³C₆-glucose labeling patterns. Empty circles indicate ¹²C, black circles denote ¹³C entering TCA cycle via pyruvate dehydrogenase (PDH), and gray circles denote ¹³C entering TCA cycle via pyruvate carboxylase (PC). (O and P) M + 2 (O) and M + 3 (P) contribution of ¹³C₆-glucose to citrate, αKG, suc, fum, and malate (n = 8). (Q and R) Pcx mRNA levels (Q, n = 3) and immunoblot detection of PC and β-actin (R). Representative image of three experiments is shown. (S) Schematic representation of [3,4]-¹³C₂-glucose labeling of TCA cycle intermediates. Empty circles indicate ¹²C, black circles denote ¹³C entering TCA cycle via PC activity. (T) M + 1 contribution of [3,4]-¹³C₂-glucose to citrate, malate, fum, and suc. Data are means ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 versus veh-treated cells (paired two-tailed Student’s t test), ^$p < 0.05 (two-way ANOVA). See also Figure S3.

Figure 3

Extracellular pyruvate and MDH2 control NAD⁺ regeneration (A) Fractional contribution of ¹³C₃-pyruvate to lactate, malate, and citrate in cells treated with vehicle (veh) or 10 μM AMA for 3 days (n = 4). (B–E) Fractional contribution of ¹³C₆-glucose to lactate (B, n = 8), malate (C, n = 8), alanine (D, n = 8) and glycerol-3-phosphate (glycerol-3-P; E, n = 8) in cells cultured with or without 1 mM pyruvate. (F) Glycerol-3-P levels in cells cultured with or without pyruvate (n = 8). (G) NAD⁺ levels in cells cultured in either full medium (1 mM pyruvate, 0.22 mM aspartate) or without pyruvate, aspartate, or combination (n = 4). (H) Flow-cytometry analysis of BrdU⁺ cells cultured in either full medium or without pyruvate, aspartate, or combination (n = 4). (I) Fractional contribution of ¹³C₄-aspartate to malate, fumarate (fum), succinate (suc), and α-ketoglutarate (αKG) after treatment for 3 days (n = 4). (J) Schematic representation of ¹³C₄-aspartate labeling patterns. Empty circles indicate ¹²C, black circles denote ¹³C labeling pattern with conversion of oxaloacetate to malate, and gray circles denote ¹³C labeling pattern with conversion of oxaloacetate to citrate. (K) M + 2 and M + 4 contribution of ¹³C₄-aspartate to suc (n = 4). (L) NAD⁺ levels in cells cultured in full medium or without pyruvate and aspartate (Pyr/Asp), supplemented or not with 1 mM α-ketobutyrate (AKB) (n = 4). (M) Flow-cytometry analysis of BrdU⁺ cells treated for 24 h with veh or AMA and cultured in full or no Pyr/Asp medium, supplemented or not with AKB (n = 4). (N and O) Malate dehydrogenase2 (Mdh2) mRNA levels (N, n = 4) and immunoblot detection of MDH2 and β-actin (O) in cells transduced with control (Mdh2^CO) or Mdh2-specific shRNA (Mdh2^KD). Representative image of three experiments is shown. (P) NAD⁺ levels in veh- and AMA-treated Mdh2^CO and Mdh2^KD cells cultured with or without pyruvate for 3 days (n = 4). (Q) Flow-cytometry analysis of BrdU⁺Mdh2^CO and Mdh2^KD cells treated for 24 h with veh or AMA and cultured with or without pyruvate (n = 4). Data are means ± SD. ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 versus veh-treated cells (paired two-tailed Student’s t test). ^$p < 0.05 (two-way ANOVA), ^§p < 0.05 (three-way ANOVA). See also Figures S4–S6.

Figure 4

Metabolic changes increase TET activity and preserve SSPCs (A and B) Ratio of α-ketoglutarate (αKG) to succinate (suc) and of αKG to 2-hydroxyglutarate (2HG) in cells treated with vehicle (veh) or 10 μM AMA for 3 days (A, n = 15) or 3 weeks (B, n = 7). (C) Immunoblot of HIF-1α with Lamin A/C as loading control in cells treated with veh or AMA for 3 days (n = 3). IOX2, an inhibitor of HIF prolyl hydroxylase 2, was used as positive control. Representative image is shown. (D) Immunoblot of dimethylated lysine 9 of histone H3 (H3K9me2), trimethylated lysine 27 of histone H3 (H3K27me3), and dimethylated lysine 79 of histone H3 (H3K79me2), with total histone 3 (H3) and Ponceau red as loading control (n = 3). Representative image is shown. (E) Dot blot assay of 5-hydroxymethylcytosine (5hmC) in cells treated with veh or AMA for 3 days, with methylene blue staining as loading control. Representative images of four experiments are shown. (F) Percentage of 5hmC of total cytosine (n = 4). (G) Dot blot assay of 5hmC in cells treated with veh or AMA in the presence or absence of 0.5 mM ascorbic acid (Asc A) with methylene blue staining as loading control. Representative images of three experiments are shown. (H–J) Number of colonies formed (H, n = 8), and percentage of SSCs (I, n = 5) and pre-BCSPs (J, n = 5) in cells treated with veh or AMA in the presence or absence of Asc A. (K and L) αKG, succinate, and 2HG levels (K, n = 4) and ratio of αKG to succinate and of αKG to 2HG (L, n = 4) in cells treated with veh or 500 nM atpenin A5 (Atp). (M) Dot blot assay of 5hmC in veh- or Atp-treated cells with methylene blue staining as loading control. Representative images of four experiments are shown. (N–P) Number of colonies formed (N, n = 6), and percentage of SSCs (O, n = 5) and pre-BCSPs (P, n = 5) in cells treated with veh (control) or Atp. (Q) Tet1, Tet2, and Tet3 mRNA levels in periosteal cells (n = 9). (R) Tet2 mRNA levels in control (Tet2^CO) or Tet2 knockdown (Tet2^KD) periosteal cells (n = 3). (S) Dot blot assay of 5hmC in Tet2^CO and Tet2^KD cells with methylene blue staining as loading control. Representative images of three experiments are shown. (T–V) Number of colonies formed (T, n = 6–7), and percentage of SSCs (U, n = 5–6) and pre-BCSPs (V, n = 5–6) in Tet2^CO and Tet2^KD cells. Data are means ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 versus veh (two-tailed Student’s t test). ^##p < 0.01 (one-way ANOVA), ^$p < 0.05 (two-way ANOVA).

Figure 5

AMA treatment induces DNA hypermethylation (A) Volcano plot showing differential methylation at CpGs in cells treated with vehicle (veh) or 10 μM AMA for 10 days (n = 3). Probes showing a significant false discovery rate of <1%. DNA hypomethylation and DNA hypermethylation are highlighted in blue and red, respectively. AMA treatment preferentially induced DNA hypermethylation (Fisher’s exact test, p < 10⁻¹⁶). (B) 5hmC estimate at CpGs of veh- or AMA-treated cells (n = 3). (C–H) Examples of AMA-induced DNA hypermethylation at CpGs located in the protein-coding gene promoters of Cxcr6 (C), Runx1 (D), Runx2 (E and F), Runx3 (G), and Wnt10b (H) (n = 3). Data are means ± SD. ^@@@p < 0.001 (Wilcoxon signed rank test with continuity correction). See also Figure S7.

Figure 6

MDH2 activity is critical for preserving SSPCs (A and B) Fumarate, succinate (suc), α-ketoglutarate (αKG), and 2-hydroxyglutarate (2HG) levels (A, n = 8) and ratios of αKG to suc and of αKG to 2HG (B, n = 8) in cells treated with vehicle (veh) or 10 μM AMA for 3 days and transduced with control (Mdh2^CO) or Mdh2-specific shRNA (Mdh2^KD). (C) Dot blot assay of 5hmC in veh- and AMA-treated Mdh2^CO and Mdh2^KD cells with methylene blue staining as loading control. Representative images of four experiments are shown. (D–F) Number of colonies formed (D, n = 4), and percentage of SSCs (E, n = 4) and pre-BCSPs (F, n = 4) in veh- and AMA-treated Mdh2^CO and Mdh2^KD cells. Data are means ± SD. ^$p < 0.05 (two-way ANOVA).

Figure 7

Periosteal cells expanded with AMA form more bone in vivo (A) H&E staining of ectopic implants, seeded with mPDCs treated with vehicle (veh) or 10 μM AMA for 3 weeks and implanted for 8 weeks (right panel is magnification of boxed area). b, bone; f, fibrous tissue; g, scaffold granule (n = 8). (B) Quantification of the bone volume formed (n = 8). (C and D) Osterix and Hoechst (DNA) staining of ectopic implants seeded with β-actin-GFP⁺ periosteal cells and implanted for 8 weeks (C, n = 4) with quantification of the percentage of β-actin-GFP⁺ cells within the Osterix⁺ population (D, n = 4). Magnifications of the boxed area show Osterix staining or merged pictures; white arrows point at β-actin-GFP⁺/Osterix⁺ cells. (E) Viability of mPDCs, labeled prior to implantation with CellTracker Green CMFDA. Viability of CMFDA⁺ cells was determined after implantation for 3 days by annexin V-propidium iodide flow cytometry (n = 4). (F–H) CD31 staining of ectopic implants, seeded with mPDCs and implanted for 8 weeks (F; right panel is magnification of boxed area; scale bars, 200 μm; n = 8), with quantification of the number of blood vessels per mm² (G) and blood vessel surface area (H) (n = 8). (I and J) Visualization (I, methylene blue staining) and quantification of colonies formed by CMFDA⁺/PI⁻ cells isolated 3 days after implantation and cultured for 1 week (J, n = 6). Data are means ± SD. ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 versus veh (unpaired two-tailed Student’s t test). Scale bars, 200 μm (A, C) and 1 cm (I).