Relationships between Slc1a5 and Osteoclastogenesis

Abstract

Slc1a5 (ASCT2) encodes a small neutral amino-acid exchanger and is the most well-studied glutamine transporter in cancer cells. To investigate the role of Slc1a5 in osteoclastogenesis, we developed Slc1a5-deficient mice by using a conventional gene-targeting approach. The Slc1a5^-/- mice showed no obvious abnormalities in growth. Glutamine uptake was assessed in Slc1a5^+/+ and Slc1a5^-/- bone marrow cells stimulated with RANKL. The rate of glutamine uptake in Slc1a5^-/- bone marrow cells was reduced to 70% of that of cells from Slc1a5^+/+ bone marrow. To confirm the involvement of Slc1a5 in osteoclast formation, bone marrow cells derived from Slc1a5^+/+ or Slc1a5^-/- mice were stimulated with RANKL and macrophage colony-stimulating factor and stained with tartrate-resistant acid phosphatase. The bone resorption activity and actin ring formation of stimulated cells were measured. The formation of multinucleated osteoclasts in bone marrow cells isolated from Slc1a5^-/- mice was severely impaired compared with those from Slc1a5^+/+ mice. RANKL-induced expression of ERK, NFκB, p70S6K, and NFATc1 was suppressed in Slc1a5^-/- osteoclasts. These results show that Slc1a5 plays an important role in osteoclast formation.

Figure 1.

Targeted disruption of Slc1a5. (A) Targeting strategy via homologous recombination in embryonic stem cells. The structures of the wild-type Slc1a5 (top), targeting vector (second), neo allele (third), and knockout allele (bottom) are shown. (B) Southern blot analysis of recombinant embryonic stem clones: genomic DNA was digested with HindIII and labeled with the 5′ and 3′ external probes.

Figure 2.

(A) PCR-based genotype analysis of mice. See Figure 1 A for primer sites. (B) RT-PCR analysis of RNA isolated from bone marrow cells. The expression of Slc1a5 was undetectable in the bone marrow cells from Slc1a5^−/− mice. The sequences for the Hprt primers are: primer 185, 5′ GTA ATG ATC AGT CAA CGG GGG AC 3′, and primer 186, 5′ CCA GCA AGC TTG CAA CCT TAA CCA 3′. The numbered black boxes indicate exons in Slc1a5.

Figure 3.

Glutamine uptake analysis in Na⁺-containing or Na⁺-free incubation buffer. (A) Glutamine uptake assays were performed in bone marrow cells stimulated with mCSF for 3 d. (B) Glutamine uptake in bone marrow cells incubated with mCSF for 3 d and then with mCSF and RANKL for 1 d. Glutamine uptake in the incubated cells derived from Slc1a5^+/+ and Slc1a5^−/− mice is normalized to protein content (in milligrams). †, P < 0.01 between Slc1a5^+/+ mice and Slc1a5^−/− mice (n = 6 per group).

Figure 4.

(A) Bone marrow cells were stimulated by mCSF and RANKL for 5 d. Cells were fixed and TRAP-stained. TRAP-positive areas were calculated by using the color extraction system of Hybrid Cell Count (Keyence, Osaka, Japan). (B) Actin ring formation. The fixed cells were stained with rhodamine phalloidin and DAPI. (C) Resorption pit formation. The bone slices were stained with 1% toluidine blue. (D) NFATc1 proteins were immunoblotted; actin served as a loading control. *, P < 0.05 between Slc1a5^+/+ mice and Slc1a5^−/− mice (n = 6 per group).

Figure 5.

GeneChip analysis of Slc1a5^+/+ (W) and Slc1a5^−/− (KO) mice. Bone marrow–derived macrophages were prepared from bone marrow cells. After the bone marrow cells were cultured in the presence of mCSF for 3 d, and the bone marrow–derived macrophages among the adherent cells were collected. Osteoclasts were generated by culturing bone marrow–derived macrophages with mCSF and RANKL for 3 d. Gene expression was assayed on an Agilent gene expression system. The values are the ratio of change related to osteogenesis and glutamine metabolism genes in bone marrow–derived macrophages compared with osteoclasts. Each graph shows osteoclast related genes (A), glutamine transporter genes (B), glutamine metabolism genes (C), amino acid depletion genes (D), fusion factor genes (E), and target genes of ERK (F). *, P < 0.05; †, P < 0.01 between Slc1a5^+/+ mice and Slc1a5^−/− mice (n = 4 per group).

Figure 6.

Immunoblotting analysis of signal events in Slc1a5^−/− bone marrow–derived macrophages. Bone marrow–derived macrophages were cultured at various time points between 0 and 60 min after treatment with RANKL. Western blotting was performed by using the antibodies indicated, and protein levels were quantified by using NIH Image. *, P < 0.05; †, P < 0.01 between Slc1a5 ^+/+ mice and Slc1a5^−/− mice (n = 5 per group).

Figure 7.

Signaling scheme of Slc1a5-mediated regulation in RANKL-induced osteoclast formation.