Research ArticleBone

ATRAID regulates the action of nitrogen-containing bisphosphonates on bone

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Science Translational Medicine  20 May 2020:
Vol. 12, Issue 544, eaav9166
DOI: 10.1126/scitranslmed.aav9166
  • Fig. 1 ATRAID is required for molecular responses to nitrogen-containing bisphosphonates (N-BPs).

    (A) Schematic of haploid mutagenesis screening pipeline. Sequencing-based identification of gene-trap insertion sites in alendronate-resistant human haploid KBM7 cells. Genomic DNA for sequencing was obtained from mutagenized KBM7 cells grown for 4 weeks after treatment with alendronate (165 μM). (B) Sequencing-based identification of gene-trap insertion sites in alendronate-resistant cells. N, number of unique insertions within the stated gene locus. False discovery rate (FDR)–corrected P values for ATRAID = 7.02 × 10−45, PLCL1 = 1.02 × 10−04, EPHB1 = 2.05 × 10−04, SNTG1 = 1.84 × 10−03. P values represent enrichment in alendronate-treated versus vehicle treated cells. (C) Schematic representation of structural features of human ATRAID protein and its mouse and frog orthologs. (D) Cell viability in wild-type control and ATRAID-deficient cells exogenously expressing or not expressing ATRAID cDNA. Cells were treated with alendronate (60 μM) and analyzed for cell viability. Cell viability was determined by measuring cellular ATP and is expressed as a ratio of that compared with untreated cells. Error bars indicate the SD for n = 4 (biological replicates). N.S., not significant; *P < 0.05, Student’s t test. v2, variant 2 (NM_080592.3); v3, variant 3 (NM_001170795.1) of the ATRAID gene, respectively. (E) Chemical structures for nitrogen-containing bisphosphonates (N-BPs) or non–N-BPs. (F) KBM7 cell viability in ATRAID-deficient (ATRAID_GT1 and ATRAID_GT2) and control (wild-type) KBM7 cells upon treatment with N-BPs or non–N-BPs. All cells were treated with the indicated concentration of the indicated N-BP (alendronate, zoledronate), BP (etidronate, tiludronate) for 72 hours. Cell viability was determined by measuring cellular ATP and is expressed as a ratio of that compared with untreated cells. All measurements were performed in quadruplicate (biological replicates). *P < 0.05, Student’s t test. (G) Immunoblots of cell lysates from ATRAID-deficient and ATRAID v3–reconstituted HEK-293T cells treated with the indicated dose of alendronate for 24 hours. Equal amounts of protein were loaded in each lane. This experiment was repeated three times (biological replicates) and was consistent all three times. Asterisk (*) indicates nonspecific band. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 2 Atraid is required for organismal responses to N-BPs.

    (A) Schematic of mouse menopausal bone loss model—bilateral ovariectomy (OVX). Saline or alendronate (100 μg/kg per week) was administered concurrent with OVX or a sham procedure. After 4 weeks, mice were euthanized and bones and serum were extracted and analyzed. (B) Representative μCT reconstructions of femoral trabecular bone from 4-month-old litter-matched derived, wild-type, Atraid WT (+/+) and KO (−/−) female mice that were either ovariectomized (OVX) or sham operated (Sham), treated with either vehicle (saline), or alendronate for 4 weeks. (C to F) Ovariectomized WT and Atraid KO mice and their bone microstructural responses to alendronate. Femur cortical (C and D) and trabeculae (E and F) regions were analyzed by μCT. Each circle represents an individual animal. Circles offset to the right represent unique animals with similar values to those of another animal (offset for visual clarity). N = 6 to 11 mice (3.5 month old) per group. *P < 0.01, # indicates 0.01 < P < 0.05, Student’s t test, and red lines indicate mean. (G and H) Ovariectomized WT and Atraid KO mice and their bone strength responses to alendronate. Stiffness (G) and yield load (H) were analyzed by three-point bending test. Each circle represents an individual animal. Circles offset to the right represent unique animals with similar values to those of another animal (offset for visual clarity). N = 6 to 11 mice per group. *P < 0.01, #0.01 < P < 0.05, Student’s t test, and red lines indicate mean.

  • Fig. 3 Atraid is required cell autonomously for N-BP inhibition of osteoclast function.

    (A) CTX-I, a serum marker of osteoclast activity, was measured in WT and AtraidKO ovariectomized mice with or without alendronate treatment by ELISA. Each circle represents an individual animal. Circles offset to the right represent unique animals with similar values to those of another animal (offset for visual clarity). N = 8 to 13 mice per group. *P < 0.05, Student’s t test. (B) Osteoclast histomorphometric responses in WT and AtraidKO ovariectomized mice with or without alendronate treatment. Osteoclast surface to bone surface ratio (Oc.S/BS) was determined by tartrate-resistant acid phosphatase (TRAP)–assay reactivity. Each circle represents an individual animal. Circles offset to the right represent unique animals with similar values to those of another animal (offset for visual clarity). N = 5 to 7 mice per group. *P < 0.05, Student’s t test, and red lines indicate mean. (C) Quantitative polymerase chain reaction (PCR) to examine mRNA expression of markers of osteoclast differentiation, Ctsk, Tnfrsf11a (RANK), Acp5 (TRAP), in wild-type (WT) and AtraidKO M-CSF–expanded bone marrow macrophages (BMMs) differentiated with RANKL to osteoclasts. Expression is normalized to wild-type, undifferentiated BMM cells, using Actb and Rplp0 as control genes. Error bars represent the SD of technical triplicate reactions. (D) Percent of Annexin-V–positive cells after a 48-hour alendronate treatment of WT and AtraidKO BMMs differentiated into osteoclasts. Annexin V staining was assessed using flow cytometry. Each circle represents osteoclasts derived from an individual animal (split for treatment with 0, 10, and 30 μM alendronate). Red lines indicate mean. *P < 0.05, Student’s t test. (E) Percent of Annexin-V–positive cells after a 48-hour alendronate treatment (0, 30, and 80 μM) in wild-type and AtraidKO differentiated RAW 264.7 osteoclasts. Annexin V staining was assessed using flow cytometry. Error bars represent the SD of n = 3 experiments (biological replicates), *P < 0.05, Student’s t test. (F) Immunoblots of cell lysates of RAW wild-type (WT) and AtraidKO (KO) cells, and RAW 264.7–derived osteoclasts treated with alendronate for 48 hours. (Top) Immunoblot specific to the unprenylated version of Rap1a. (Bottom) Gapdh, serving as a loading control. Alendronate concentrations were 0, 20, and 80 μM. (G) Representative image of a six-well dish coculture of equal numbers of mouse primary osteoblasts and osteoclasts of the indicated genotypes with or without the indicated doses of alendronate for 4 days. The experiment was performed three independent times with a similar result. Red staining reflects TRAP-assay reactivity. (H) Image analysis of the samples in (G). Error bars represent the SD of n = 3 independent images (technical replicates). *P < 0.01, Student’s t test.

  • Fig. 4 ATRAID as a potential genetic factor for altered responses to N-BPs in patients.

    (A) Genome-wide studies of N-BP responsiveness in patients versus cells. The outcomes considered from human studies involving N-BPs are osteonecrosis of the jaw (ONJ), breast cancer bone marrow micrometastases [disseminated tumor cells (DTCs)], and atypical femoral fractures (AFFs). ATRAID, ATR, and ZBTB4 are statistically significant hits (FDR corrected P < 0.05) in N-BP cell–based CRISPRi/a screening, differentially expressed in both gene expression datasets (ONJ and DTCs), and have rare multiple nonsynonymous coding variants in AFF and ONJ cases but not controls. These three genes are visualized as the Venn diagram of overlap of lists of genes that met the following criteria: significant alendronate CRISPRi, zoledronate CRISPRi, or alendronate CRISPRa hits with absolute value of rho growth phenotype values ≥ 0.30 and P ≤ 0.05 (1335 of 15828 genes) (12, 38); differentially expressed N-BP in ONJ + DTCs [774 of 18415 for ONJ (36) and 20492 for DTCs (37)]; multiple coding variant(s) in AFF and ONJ cases and not controls (1252 of 11,659 genes) (data as part of this study). (B) Patient genetic data for ATRAID. Raw expression values were normalized to 1 to fit on a comparable y axis. *P < 0.05, moderated t test. “x”-enriched refers to the fold-enrichment of the allele compared with a population with a similar genetic background as the cases. For example, for ATRAID, the D5G variant is present in 2 of 27 patients with AFF. Although this allele was not detected in the 11 control samples, it is present in a population of European Americans (EA) and Asian Americans (AA) that is representative of the study population at a prevalence of 0.0131. Therefore, the D5G allele is (2/27)/0.0131 = 5.66X enriched in cases compared to the EA/AA population. “v3” and “v2” refer to isoforms of the ATRAID gene. A simple binomial test was used to calculate the significance of each variant. * P < 0.05. (C) Quantitative PCR to examine ATRAID mRNA expression in wild-type, ATRAID-deficient, and low ATRAID-expressing cells. Error bars represent the SD of technical triplicate reactions. Expression was normalized to WT cells using RPLP0 and TBP as controls. *P < 0.05, Student’s t test. (D) Cell viability in wild-type, ATRAID-deficient, and low ATRAID-expressing cells. Cells were treated with the indicated doses of alendronate and analyzed for cell viability. Cell viability was determined by measuring cellular ATP and is expressed as a ratio of that compared with untreated cells. Error bars indicate the SD for n = 4 (biological replicates). * and # indicate P < 0.05 for the indicated cell lines, Student’s t test. (E) Immunoblot (IB) of wild-type and D5G/G32R variant ATRAID-V5–tagged proteins. Mutant or wild-type ATRAID v2 and v3 were stably introduced into ATRAID-deficient HEK-293T cells. (F) Cell viability in wild-type versus D5G/G32R variant cells. Cells were treated with the indicated doses of alendronate and analyzed for cell viability. Cell viability was determined by measuring cellular ATP and is expressed as a ratio of that compared with untreated cells. Error bars indicate the SD for n = 4 (biological replicates). *P < 0.05, Student’s t test.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/544/eaav9166/DC1

    Materials and Methods

    Fig. S1. ATRAID is required for the cellular responses to N-BPs.

    Fig. S2. Generation and skeletal characterization of AtraidKO mice.

    Fig. S3. Atraid is required cell autonomously for the effects of N-BP on osteoclasts in two models of osteoporosis.

    Data file S1. Results of haploid genomic screen for genes required for the response to alendronate.

    Data file S2. Statistics for AtraidKO mice basal characterization and statistics for bone structure and strength of ovariectomized wild-type and AtraidKO animals treated with alendronate.

    Data file S3. Statistics for bone histomorphometry and serum bone proteins in ovariectomized and senile wild-type and AtraidKO animals treated with alendronate.

    Data file S4. Gene expression, sequencing, and cell growth phenotype data for ONJ, DTCs, AFF, and CRISPRi/a studies.

    References (5281)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. ATRAID is required for the cellular responses to N-BPs.
    • Fig. S2. Generation and skeletal characterization of AtraidKO mice.
    • Fig. S3. Atraid is required cell autonomously for the effects of N-BP on osteoclasts in two models of osteoporosis.
    • Legends for data files S1 to S4
    • References (5281)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Results of haploid genomic screen for genes required for the response to alendronate.
    • Data file S2 (Microsoft Excel format). Statistics for AtraidKO mice basal characterization and statistics for bone structure and strength of ovariectomized wild-type and AtraidKO animals treated with alendronate.
    • Data file S3 (Microsoft Excel format). Statistics for bone histomorphometry and serum bone proteins in ovariectomized and senile wild-type and AtraidKO animals treated with alendronate.
    • Data file S4 (Microsoft Excel format). Gene expression, sequencing, and cell growth phenotype data for ONJ, DTC, AFF, and CRISPRi/a studies.

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