Notch signaling suppresses glucose metabolism in mesenchymal progenitors to restrict osteoblast differentiation

Abstract

Notch signaling critically controls cell fate decisions in mammals, both during embryogenesis and in adults. In the skeleton, Notch suppresses osteoblast differentiation and sustains bone marrow mesenchymal progenitors during postnatal life. Stabilizing mutations of Notch2 cause Hajdu-Cheney syndrome, which is characterized by early-onset osteoporosis in humans, but the mechanism whereby Notch inhibits bone accretion is not fully understood. Here, we report that activation of Notch signaling by either Jagged1 or the Notch2 intracellular domain suppresses glucose metabolism and osteoblast differentiation in primary cultures of bone marrow mesenchymal progenitors. Importantly, deletion of Notch2 in the limb mesenchyme increases both glycolysis and bone formation in the long bones of postnatal mice, whereas pharmacological reduction of glycolysis abrogates excessive bone formation. Mechanistically, Notch reduces the expression of glycolytic and mitochondrial complex I genes, resulting in a decrease in mitochondrial respiration, superoxide production, and AMPK activity. Forced activation of AMPK restores glycolysis in the face of Notch signaling. Thus, suppression of glucose metabolism contributes to the mechanism, whereby Notch restricts osteoblastogenesis from bone marrow mesenchymal progenitors.

Keywords: Bone Biology; Glucose metabolism.

Figure 1. Notch signaling suppresses glycolysis in bone marrow mesenchymal progenitors.

(A) Relative mRNA levels of the indicated genes assayed by RT-qPCR in R26-NICD2 BMSCs infected with Ad-GFP or Ad-Cre and treated with mineralization media for 48 hours. n = 3. (B) AP or von Kossa staining in R26-NICD2 BMSCs infected with Ad-GFP or Ad-Cre after 4 days or 2 weeks, respectively, in mineralization media. (C) Glucose consumption and lactate production by R26-NICD2 BMSCs infected with Ad-GFP or Ad-CRE in regular growth media for 48 hours. n = 3. (D) Diagram of glycolysis and its key enzymes. (E) Western blots in R26-NICD2 BMSCs infected with Ad-GFP or Ad-CRE for 24 or 48 hours. Protein levels were normalized to β-actin and designated 1 in samples infected with Ad-GFP. Quantification (mean ± SD) was determined from 3 independent samples. (F) Glucose consumption and lactate production from WT BMSCs with or without Jagged1 stimulation for 48 hours. n = 3. (G) Western blots in WT BMSCs with or without Jagged1 stimulation for the indicated durations. Protein levels were normalized to β-actin or tubulin, and quantification (mean ± SD) was determined from 3 independent samples. *P < 0.05, by 2-tailed Student’s t test (A, C, and E–G). Hk2: hexokinase 2; Pfkfb3/4: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 or 4; F-2,6-P: fructose 2,6-bisphosphate; Pfk1: phosphofructokinase 1; Eno: enolase; Ldha: lactate dehydrogenase a; Pkm: pyruvate kinase, muscle.

Figure 2. Suppression of glycolysis abolishes excessive bone formation caused by Notch2 deletion.

(A) Western blots of long bones from 8-week-old Prx1-Cre Notch2^fl/fl (PN2) mice and Notch2^fl/fl (N2) littermates. Each lane is for a separate mouse. (B) Glucose consumption and lactate production in N2 and PN2 BMSCs with or without Jagged1 treatment for 48 hours. n = 3. (C–I) Analyses of 7-week-old N2 or PN2 mice that received 3PO or DMSO for 4 weeks starting at 3 weeks of age, including body weight (C), blood glucose (D), ¹⁴C-2DG uptake in bone (E), μCT images (F), μCT quantification (G), and dynamic histomorphometry (H) of trabecular bone in the proximal tibia, as well as serum CTX-I levels (I). n = 3. *P < 0.05, by 2-way ANOVA followed by Bonferroni’s post hoc test (C–E, H, and I) or 2-tailed Student’s t test (B and G). BV, bone volume; DPM, disintegrations per minute; TV, total volume.

Figure 3. Notch2 signaling suppresses glycolysis in ST2 cells.

(A) Glucose consumption and lactate production by GFP-ST2 or NICD2-ST2 cells treated with vehicle or Dox for 48 hours. n = 3. (B–D) Seahorse analyses of NICD2-ST2 cells treated with vehicle or Dox for 48 hours. n = 10. (E) Relative ATP levels in NICD2-ST2 cells treated with vehicle or Dox for 48 hours. n = 6. (F) Glucose uptake assays in NICD2-ST2 cells treated with vehicle or Dox for the indicated durations. n = 6. (G) Western blots of NICD2-ST2 cells treated with vehicle or Dox for the indicated durations. β-Actin was used for normalization and quantification (mean ± SD) was determined from 3 independent samples. After normalization to β-Actin, the levels in “-Dox” lanes were designated 1 and those in the “+Dox” lanes were further normalized. *P < 0.05, by 2-tailed Student’s t test (A and D–G).

Figure 4. Canonical Notch signaling suppresses glycolysis.

(A–C) Effects of retroviral expression of GFP or dnMaml1 on glucose consumption (A), lactate production (B) or metabolic enzymes (C) in NICD2-ST2 cells treated with vehicle or Dox for 48 hours. β-Actin was used for normalization, and quantification (mean ± SD) was determined from 3 independent samples. After normalization to β-Actin, the level in the left-most lane is designated 1, and those of the other lanes are further normalized to the left-most lane. (D) Glucose consumption and lactate production in Hey1-ST2 cells treated with vehicle or Dox for 48 hours. Western blot shows the induction of Flag-Hey1 by Dox. n = 3. (E) Relative mRNA levels of NFATc1 by RT-qPCR in Hey1-ST2 cells treated with vehicle or Dox for 48 hours. n = 3. (F) RT-qPCR analyses of Hey1 in NICD2-ST2 cells infected with lentivirus expressing 2 different Hey1 shRNAs or the control luciferase shRNA (shLuc). (G) Glucose consumption and lactate production in NICD2-ST2 cells infected with lentivirus expressing different shRNAs, with or without Dox for 48 hours. (H) Relative glucose consumption in NICD2-ST2 cells infected with lentivirus expressing different shRNAs, with or without Dox for 48 hours. Glucose consumption was normalized to the protein amount and then to the vehicle-treated group designated as 1. (I) RT-qPCR analyses of mRNA levels showing shRNA knockdown efficiency. n = 3. *P = 0.05, by 2-way ANOVA followed by Bonferroni’s post hoc test (A, B, F, and I) or 2-tailed Student’s t test (C, E, G, and H).

Figure 5. Canonical Notch signaling suppresses gene expression of glycolytic enzymes and components of the mitochondrial ETC.

(A and B) Relative mRNA levels of glycolytic enzymes (A) and components of the mitochondrial ETC (B) in NICD2-ST2 cells with retroviral expression of GFP or dnMaml1 and treatment with vehicle or Dox for 24 hours. (C) Relative glucose consumption in NICD2-ST2 cells infected with lentivirus expressing different shRNAs, as indicated, with or without Dox for 48 hours. Glucose consumption was normalized to the protein amount and then to the no-Dox control designated as 1. n = 3. *P < 0.05, by 2-way ANOVA followed by Bonferroni’s post hoc test (A and B) or 2-tailed Student’s t test (C).

Figure 6. Notch suppresses glycolysis through downregulation of AMPK activity.

(A–C) Measurements of mitochondrial complex I activity (n = 4) (A), mitochondrial superoxide levels (n = 6) (B), and p-AMPKα levels (n = 3) (C) in NICD2-ST2 cells treated with vehicle or Dox for the indicated durations. Total AMPKα was used for normalization, and quantification (mean ± SD) was determined from 3 independent samples. After normalization to total AMPKα, the level in the left lane was designated 1 and that of the right lane was further normalized to the left lane. mOD, mitochondrial OD. (D and E) Western blot analyses of NICD2-ST2 cells with retroviral expression of GFP or dnMaml1 and treatment with vehicle or Dox for 24 hours. Phosphorylation levels were normalized to total AMPKα or ACC, and quantification (mean ± SD) was determined from 3 independent samples. After normalization to total protein, the level in the left-most lane was designated 1 and the other lanes were further normalized to that lane. (F) Western blots of BMSCs with or without Jagged1 stimulation for 24 hours. Normalization and quantification were determined as in D and E. (G) Effects of AICAR at the indicated concentration for 48 hours on glucose consumption and lactate production in BMSCs with or without Jagged1 stimulation. n = 3. *P < 0.05, by 2-tailed Student’s t test (A–G).

Figure 7. AMPK activation rescues metabolic inhibition by Notch2 without restoring glycolytic gene expression.

(A) Effects of AICAR for 48 hours on glucose consumption and lactate production in NICD2-ST2 cells. (B) RT-qPCR analyses of relative mRNA levels of glycolytic genes. (C) Western blot and RT-qPCR analyses of ST2 cells infected with lentivirus expressing shRNAs targeting luciferase (Luc) or Ndufaf2. pAMPK was first normalized to total AMPK in each lane. The normalized level in the left -most lane was designated 1 and those of the other lanes were further normalized to that lane. All RT-qPCR data were normalized to 18S rRNA. n = 3. *P < 0.05, by 2-way ANOVA followed by Bonferroni’s post hoc test (A) or 2-tailed Student’s t test (B and C).

Figure 8. Working model for Notch signaling in the suppression of glucose metabolism in mesenchymal progenitors.

Canonical Notch signaling induces multiple transcriptional repressors that suppress the transcription of genes encoding glycolytic enzymes or mitochondrial complex I components. The decrease in mitochondrial respiration lowers AMPK activity and helps to reduce glycolysis.