Research ArticleBone Biology

The L-type amino acid transporter LAT1 inhibits osteoclastogenesis and maintains bone homeostasis through the mTORC1 pathway

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Science Signaling  09 Jul 2019:
Vol. 12, Issue 589, eaaw3921
DOI: 10.1126/scisignal.aaw3921
  • Fig. 1 A LAT-dependent amino acid uptake system operates in both osteoclasts and osteoblasts.

    (A) Schematic diagram of the procedure for generating [125I]IMT. (B) Primary osteoblasts and osteoclasts cultured from wild-type (WT) mice were incubated with [125I]IMT at 4° or 37°C for 30 min in HBSS buffer. n = 4 cell cultures from different mice. (C to F) Primary cells were incubated with [125I]IMT at 37°C for 30 min in HBSS buffer in which NaCl was replaced with equimolar ChoCl (C and D) or in HBSS buffer containing JPH203 (E and F). n = 4 cell cultures from different mice. Data were analyzed by the two-tailed Student’s t test. *P < 0.05 and **P < 0.01, significantly different from the value obtained in cells incubated at 4°C (B), cells incubated in HBSS buffer with NaCl (C and D), or cells incubated in HBSS buffer with dimethyl sulfoxide (DMSO) (E and F).

  • Fig. 2 Slc7a5 expression in preosteoclasts is reduced in ovariectomized mice.

    (A) C57BL6 mice were subjected to ovariectomy (OVX) or sham operation and subsequent isolation of CD11blow/−CX3CR1+Ly6Chi cells (preosteoclasts) and CD45CD51+Sca1 cells (osteoblasts) by flow cytometry. (B and C) Quantification of mRNAs encoding amino acid transporters in preosteoclasts (B) and osteoblasts (C) by qPCR. n = 5 mice. All data were analyzed by the two-tailed Student’s t test. **P < 0.01, significantly different from the value obtained for sham-operated mice. U.D., under detectable values.

  • Fig. 3 Slc7a5 in osteoclasts is essential for bone resorption and bone homeostasis in vivo.

    (A) Schematic diagram of the generation of osteoclast-specific Slc7a5 knockout mice using the osteoclast-specific Cre drivers Tnfrs11a and Lyz2. (B) Slc7a5 expression in cultured osteoclasts from Slc7a5fl/fl (control) and Tnfrsf11a-Cre;Slc7a5fl/fl mice as determined by PCR. Actb is a control. n = 3 mice per genotype. (C) μCT analysis and (D) bone volume–to–tissue volume (BV/TV) ratio measurements of femurs from Slc7a5fl/fl and Tnfrsf11a-Cre;Slc7a5fl/fl mice. (E) von Kossa staining, (F) BV/TV, (G) number of osteoblasts/tissue area (N.Ob/T.Ar), (H) bone formation rate (BFR), and (I) osteoclast surface/bone surface (Oc.S/BS) measurements from vertebrae and (J) P1NP and (K) CTx concentrations in serum of Slc7a5fl/fl and Tnfrsf11a-Cre;Slc7a5fl/fl mice at 12 weeks of age. n = 5 to 7 Slc7a5fl/fl mice; n = 6 to 9 Tnfrsf11a-Cre;Slc7a5fl/fl mice. (L) von Kossa staining, (M) BV/TV, and (N) Oc.S/BS of vertebrae and (O) P1NP and (P) CTx concentrations in serum from Slc7a5fl/fl and Lyz2-Cre;Slc7a5fl/fl mice at 12 weeks of age. n = 7 Slc7a5fl/fl mice; n = 8 Lyz2-Cre;Slc7a5fl/fl mice. All data were analyzed by the two-tailed Student’s t test. *P < 0.05 and **P < 0.01, significantly different from the value obtained for control mice. Scale bars, 1 mm (C) and 200 μm (E and L).

  • Fig. 4 Slc7a5 in osteoblasts is dispensable for bone formation and bone homeostasis in vivo.

    (A) Schematic diagram of the generation of osteoblast-specific Slc7a5 knockout mice. (B) Slc7a5 expression in the GFP-positive cells of control and Osx-Cre;Slc7a5fl/fl mice as determined by PCR. n = 3 mice per genotype. (C) μCT analysis, (D) BV/TV ratio as determined by μCT, (E) von Kossa staining, (F) BV/TV as determined by von Kossa staining, (G) TRAP staining, (H) Oc.S/BS ratio, (I) calcein labeling, (J) BFR, and (K) N.Ob/T.Ar of femurs from control and Osx-Cre;Slc7a5fl/fl mice at 12 weeks of age. n = 6 Osx-Cre mice; n = 8 Osx-Cre;Slc7a5fl/fl mice. All data were analyzed by the two-tailed Student’s t test. The double-headed white arrow in (I) indicates the distance between calcein double labeling. Scale bars, 1 mm (C and E), 100 μm (G), and 10 μm (I).

  • Fig. 5 Slc7a5 deficiency stimulates osteoclastogenesis in vitro.

    BMMs from Slc7a5fl/fl mice were retrovirally infected with Cre recombinase and subsequently stimulated with RANKL to induce osteoclast differentiation. Cells were then analyzed for (A to C) the number of TRAP-positive multinucleated cells MNCs and pit formation area, (D) expression of osteoclast marker genes, (E) cell proliferation ratio, and (F) cell survival. n = 4 to 6 mice per genotype. Data were analyzed by the two-tailed Student’s t test (B to D) or the two-way ANOVA with Bonferroni and/or Dunnett post hoc test (E and F). *P < 0.05 and **P < 0.01, significantly different from the value obtained in control cells. Arrows in (A) indicate representative resorption pits. Scale bar, 50 μm.

  • Fig. 6 mTORC1 activation rescues Slc7a5 deficiency–induced osteoclast activation in vitro and in vivo.

    (A to C) BMMs from Slc7a5fl/fl mice were retrovirally infected with Cre recombinase and subsequently stimulated with RANKL, followed by immunoblotting and densitometric quantification of phosphorylation of the indicated downstream effectors of mTORC1 signaling. β-Actin is a loading control. n = 4 mice per genotype. (D and E) BMMs from Slc7a5fl/fl mice were retrovirally infected with Cre recombinase and constitutively active (CA) Rheb, an upstream activator of mTORC1, and subsequently treated with RANKL, followed by determination of the number of TRAP-positive multinucleated cells. n = 5 mice per genotype. E.V., empty vector “pMX-Control”. (F) μCT analysis and (G) BV/TV ratio and Oc.S/BS ratio of femurs and P1NP and CTx concentrations in serum of Tnfrsf11a-Cre;Slc7a5fl/fl;Tsc1fl/+ mice at 12 weeks of age. n = 6 control mice (Slc7a5fl/fl;Tsc1fl/+ mice or Slc7a5fl/+;Tsc1fl/+ mice) and Tnfrsf11a-Cre;Slc7a5fl/fl mice; n = 7 Tnfrsf11a-Cre;Slc7a5fl/fl;Tsc1fl/+ mice. Data were analyzed by the two-way ANOVA with Bonferroni and/or Dunnett post hoc test (B, C, and E) or the one-way ANOVA with Bonferroni and/or Dunnett post hoc test (G). *P < 0.05 and ** P < 0.01, significantly different from control cells (B, C, and E) or control mice (G). #P < 0.05 and ##P < 0.01, significantly different from the value obtained for Slc7a5-deficient cells infected with control vector (E) or Tnfrsf11a-Cre;Slc7a5fl/fl mice (G). Scale bars, 50 μm (D) and 1 mm (F).

  • Fig. 7 Nuclear accumulation of NFATc1 increases in Slc7a5-deficient osteoclasts.

    BMMs from Slc7a5fl/fl mice were retrovirally infected with Cre recombinase and subsequently stimulated with RANKL, followed by immunoblotting and densitometric analysis of (A to C) NFATc1 abundance in the nucleus and whole-cell extracts and (D to I) phosphorylation of Erk1/2, JNK1/2, and p38. n = 4 mice per genotype. All data were analyzed by the two-way ANOVA with Bonferroni and/or Dunnett post hoc test. **P < 0.01, significantly different from the value obtained in control cells.

  • Fig. 8 The Akt-GSK3β and canonical NF-κB pathways are activated in Slc7a5-deficient osteoclasts.

    BMMs from Slc7a5fl/fl mice were retrovirally infected with Cre recombinase and subsequently stimulated with RANKL, followed by immunoblotting and densitometric quantification of (A to C) phosphorylation of Akt and GSK3β, (D to F) phosphorylation of IKKα/β and IκBα, and (G and H) abundance of p65. n = 4 mice per genotype. (I and J) BMMs from Slc7a5fl/fl mice were retrovirally infected with Cre recombinase and subsequently stimulated with RANKL in the presence or absence of Akt inhibitor X (I) or the IKK inhibitor BMS-345541 (J), followed by TRAP staining. n = 4 mice per genotype. All data were analyzed by the two-way ANOVA with Bonferroni and/or Dunnett post hoc test. **P < 0.01, significantly different from the value obtained in control cells. ##P < 0.01, significantly different from the value obtained in Slc7a5-deficient cells without inhibitors. (K) Model for LAT1-mediated control of osteoclast activation. The LAT1-mTORC1 axis (red dashed square) inhibits the activation of Akt, thus preventing GSK3β from stimulating the translocation of NFATc1 from the nucleus to the cytoplasm. LAT1-mTORC1 signaling also inhibits the activation of IKK and IκBα in osteoclasts, thus preventing the degradation of IκB and the nuclear translocation of the p65 and p50 subunits that constitute NF-κB. These actions repress the nuclear accumulation of NFATc1 and Nfatc1 expression, respectively, leading to the inhibition of NFATc1-dependent osteoclastic gene expression, thereby impeding osteoclastogenesis.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/589/eaaw3921/DC1

    Fig. S1. Expression of genes encoding amino acid transporters in preosteoclasts and osteoblasts.

    Fig. S2. Expression of Slc7a5 mRNA in mutant mice.

    Fig. S3. Phosphorylation of p70S6K1 by EAA deprivation in osteoclasts.

    Fig. S4. Effect of RANKL on [125I]IMT incorporation in osteoclasts.

    Fig. S5. Phosphorylation of eIF2α in Slc7a5-deficient cells.

    Fig. S6. Effects of Akt and NF-κB inhibitors in Slc7a5-deficient cells.

    Table S1. List of primers used for real-time PCR.

  • This PDF file includes:

    • Fig. S1. Expression of genes encoding amino acid transporters in preosteoclasts and osteoblasts.
    • Fig. S2. Expression of Slc7a5 mRNA in mutant mice.
    • Fig. S3. Phosphorylation of p70S6K1 by EAA deprivation in osteoclasts.
    • Fig. S4. Effect of RANKL on [125I]IMT incorporation in osteoclasts.
    • Fig. S5. Phosphorylation of eIF2α in Slc7a5-deficient cells.
    • Fig. S6. Effects of Akt and NF-κB inhibitors in Slc7a5-deficient cells.
    • Table S1. List of primers used for real-time PCR.

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