Research ArticleBONE DISEASE

The PTH/PTHrP-SIK3 pathway affects skeletogenesis through altered mTOR signaling

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Science Translational Medicine  19 Sep 2018:
Vol. 10, Issue 459, eaat9356
DOI: 10.1126/scitranslmed.aat9356
  • Fig. 1 Homozygosity for a missense mutation in SIK3 produces a novel skeletal disorder.

    (A to F) Radiographs of R07-429A and R07-429B showing a large separation of the irregular epiphyses from metaphyses, widened/flared metaphyses with irregular ossifications and irregular ossification front (A and B), brachydactyly with irregular metacarpal and phalangeal metaphyses and delayed epiphyseal ossification (C and D), absence of pubic bone ossification (E), and delayed ossification of the basal occipital bone and subjectively increased density of the skull (F). Arrows point to different defects described. (G and H) Western blots and quantification of SIK3 protein concentrations in patient-derived fibroblasts compared to control cells. Graphs represent means ± SEM. Student’s t test, *P < 0.05. Control and R07-429A, n = 5; R07-429B, n = 3. (I) SIK3 gene expression as determined by quantitative polymerase chain reaction (qPCR). Graphs represent means ± SEM. Control and R07-429A, n = 5; R07-429B, n = 3. Individual values of quantifications are provided in table S2.

  • Fig. 2 Impaired kinase function in SIK3R129C mutant.

    (A) Alignment of SIK3 orthologs demonstrating evolutionary conservation of p.R129 (conserved residues in gray; R129 in red). (B) 3D homology model of SIK3 kinase domain with indicated adenosine 5′-triphosphate (ATP)–binding site (green), substrate binding site (yellow), and activation loop (blue); arrow indicates position of R129. (C) Magnified view of the mutant p.R129 (red) surroundings. (D) C-terminally FLAG-tagged wild-type (WT) and R129C SIK3 variants transfected into human embryonic kidney (HEK) 293 T cells and purified by FLAG IP. The quantities of three independent transfections for each SIK3 variant were determined by Western blot (WB). Cells transfected with empty plasmid were labeled empty. (E to G) Purified SIK3 in cell-free kinase assays with radioactive ATP and AMARA or CHKtide peptide substrates. Phosphorylation signal was determined by ATP[32P] autoradiography of kinase reaction spotted on blotting paper (E and F) or by scintillation (G). Recombinant active SIK3 was used as a positive control for kinase activity; samples with ATP omitted served as negative controls. (F) ATP[32P] signal shown in (E) normalized to concentration of immunoprecipitated SIK3 in each reaction. Graphs represent means ± SEM. AMARA, n = 7; CHKtide, n = 11. Student’s t test, *** P < 0.0001. (G) ATP[32P] scintillations expressed as percentages of signal obtained in kinase assays with WT SIK3. Signal in immunocomplexes isolated from cells transfected with empty plasmid instead of SIK3 (as denoted by “E,” red dashed lines) represents the background activity. Graphs represent means ± SEM. AMARA (from left to right), n = 14, n = 15, n = 6, n = 6, n = 6, n = 6, and n = 6; CHKtide (from left to right), n = 28, n = 28, n = 10, n = 10, n = 10, n = 10, and n = 10. Student’s t test, ***P < 0.0001. Individual values of quantifications are provided in table S2. C.P.M., counts per minute.

  • Fig. 3 SIK3-deficient cells show decreased mTORC1 and mTORC2 activity due to accumulation of DEPTOR.

    (A) Western blots showing amounts of indicated proteins in patient and control cells starved overnight (O/N) before protein extraction. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Co-IP of SIK3 with different mTORC1/mTORC2 components. C-terminally FLAG-tagged WT (SIK3) and R129C SIK3 variants were transfected into HEK293T cells and purified by FLAG IP. Co-IP of endogenous RICTOR, RAPTOR, and GβL was detected by Western blot. (C) C-terminally FLAG-tagged WT (SIK3), R129C SIK3 variants, and N-terminally hemagglutinin (HA)–tagged DEPTOR were cotransfected into HEK293T cells and purified by HA IP. Co-IP of SIK3 was detected by Western blot. (D) Western blots of control and patient-derived fibroblasts starved overnight and then treated with 10% fetal bovine serum (FBS) for 4 to 7 hours. (E) Western blots of control fibroblasts transfected with 10 nM small interfering RNA (siRNA) and control siRNA (control). Forty-eight hours after transfection, cells were starved overnight and then treated with 10% FBS for 4 hours or overnight. (F) N-terminally HA-tagged DEPTOR was transfected into control and patient-derived fibroblasts, and 24 hours later, cells were starved overnight before harvest or treatment with 10% serum + 15 μM MG132 for 10 hours. DEPTOR was immunopurified using HA beads, and its interaction with endogenous β-transducin repeats-containing protein (β-TRCP) was detected by Western blot. EV, empty vector (negative control). WCL, whole-cell lysates. Quantification of Western blots is shown in figs. S2 and S4. Individual values of quantifications are provided in table S2.

  • Fig. 4 SIK3 is coexpressed with mTOR components in the growth plate.

    Immunolocalization of SIK3, DEPTOR, and pS6 in P1 (A to H) and P21 (I to P) mouse femur growth plates. (A) Picrosirius red hematoxylin staining of P1 cartilage growth plate chondrocytes. (B to D) Immunolocalization of SIK3 (B), DEPTOR (C), and pS6 (D) at P1. (E to H) Magnification of boxed regions in (A) to (D). (I) Picrosirius red hematoxylin staining of P21 growth plate. (J to L) Immunolocalization of SIK3 (J), DEPTOR (K), and pS6 (L) at P21. (M to P) Magnification of boxed regions in (I) to (L). Dashed lines represent separation of proliferative and hypertrophic zones. Arrows point to zones of expression detected with the different antibodies. Scale bars, 50 [(A to H) and (M to P)] and 200 μm (I to L).

  • Fig. 5 PTH/PTHrP signaling regulates mTOR activity through SIK3-mediated DEPTOR degradation.

    (A) Western blot of JMC-derived patient and control chondrocytes starved overnight and then treated with 10% FBS for 4 hours. Quantification shown in fig. S5. Individual values of quantifications are provided in table S2. (B to E) Picrosirius red hematoxylin staining of control (B and C) and JMC patient (D and E) proximal phalange growth plates. Magnifications of boxed regions in (B) and (D) are shown in (C) and (E). (F to I) Immunolocalization of DEPTOR in control (F and H) and patient growth plates (G and I). Magnifications of boxed regions in (F) and (G) are shown in (H) and (I). Arrows indicate expression of DEPTOR in proliferative chondrocytes and absence in hypertrophic cells in control compared to constant expression throughout the growth plate in the patient. Scale bars, 50 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/459/eaat9356/DC1

    Fig. S1. Patients show a missense mutation in the catalytic domain of SIK3.

    Fig. S2. Quantification of protein amounts of Western blots in Fig. 3A.

    Fig. S3. Serum and insulin treatment in control and patient cells.

    Fig. S4. Quantification of protein amounts of Western blots in Fig. 3 (D and E).

    Fig. S5. Quantification of protein amounts of Western blots in Fig. 5.

    Fig. S6. PTHrP treatment in primary chondrocytes.

    Fig. S7. Model of action of SIK3.

    Table S1. Clinical findings of affected individuals with a mutation in SIK3.

    Table S2. Individual subject-level data.

  • The PDF file includes:

    • Fig. S1. Patients show a missense mutation in the catalytic domain of SIK3.
    • Fig. S2. Quantification of protein amounts of Western blots in Fig. 3A.
    • Fig. S3. Serum and insulin treatment in control and patient cells.
    • Fig. S4. Quantification of protein amounts of Western blots in Fig. 3 (D and E).
    • Fig. S5. Quantification of protein amounts of Western blots in Fig. 5.
    • Fig. S6. PTHrP treatment in primary chondrocytes.
    • Fig. S7. Model of action of SIK3.
    • Table S1. Clinical findings of affected individuals with a mutation in SIK3.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Individual subject-level data.

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