Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jun 28;119(26):e2201490119.
doi: 10.1073/pnas.2201490119. Epub 2022 Jun 21.

IFT80 negatively regulates osteoclast differentiation via association with Cbl-b to disrupt TRAF6 stabilization and activation

Vishwa Deepak  1 , Shu-Ting Yang  1 , Ziqing Li  1 , Xinhua Li  1 , Andrew Ng  2 , Ding Xu  2 , Yi-Ping Li  3 , Merry Jo Oursler  4   5 , Shuying Yang  1   6   7
Affiliations

IFT80 negatively regulates osteoclast differentiation via association with Cbl-b to disrupt TRAF6 stabilization and activation

Vishwa Deepak et al. Proc Natl Acad Sci U S A. .

Erratum in

Abstract

Excess bone loss due to increased osteoclastogenesis is a significant clinical problem. Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation. The role of IFT80, an IFT complex B protein, in osteoclasts (OCs) is completely unknown. Here, we demonstrate that deletion of IFT80 in the myeloid lineage led to increased OC formation and activity accompanied by severe bone loss in mice. IFT80 regulated OC formation by associating with Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) to promote protein stabilization and proteasomal degradation of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). IFT80 knockdown resulted in increased ubiquitination of Cbl-b and higher TRAF6 levels, thereby hyperactivating the receptor activator of nuclear factor-κβ (NF-κβ) ligand (RANKL) signaling axis and increased OC formation. Ectopic overexpression of IFT80 rescued osteolysis in a calvarial model of bone loss. We have thus identified a negative function of IFT80 in OCs.

Keywords: IFT80; bone; osteoblast; osteoclast.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
LysM-Cre IFT80 cKO mice are osteopenic. (A) Representative Western blot image demonstrating protein expression of IFT80 (80 kDa) in BMMs and OCs (n = 5). (B) Representative Western blot image demonstrating deletion of IFT80 (n = 5); β-actin (43 kDa). (C) μCT analysis of the femurs from 12-wk-old control and IFT80d/d mice. (Scale bars, 1 mm.) Quantitative analysis of the percentage of bone volume over total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp) in the femurs of 12-wk-old control and cKO mice (n = 5). (D) Histological sections of tibiae from 12-wk-old control and IFT80d/d mice stained for TRAP (red) (n = 5). (D, Lower) Images represent enlarged views. Blue arrowheads represent OCs derived from the blue dotted boxes (Upper). Oc.S/B.S., osteoclast surface/bone surface (n = 5). (E–G) Calcein labeling in 12-wk-old LysM and IFT80d/d mice assessed using nondecalcified frozen sections of trabecular bone (E) and its quantification (F and G) (BFR, bone formation rate; MAR, mineral apposition rate) (n = 5). (Scale bar, 100 µm.) (H) Serum P1NP levels (n = 5). (I) Serum CTX-1 levels (n = 5). (J–L) Serum OPG (J), RANKL (K), and OPG-to-RANKL ratio (L) (n = 5). Results are expressed as mean ± SD. Data were analyzed using unpaired two-tailed t test. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, nonsignificant.
Fig. 2.
Fig. 2.
Ablation of IFT80 increases OC differentiation. BMMs obtained from the long bones of LysM-Cre or IFT80d/d mice were differentiated into OCs for 5 d. (A) Number of TRAP+ stained OCs or nuclei number in OCs (n = 10). (A, Lower) Tenfold enlarged images. (B) OB/OC coculture model demonstrating increased differentiation of OCs (TRAP+) in the IFT80d/d group cultured with WT OBs (n = 8). (C) Increased acidification as seen by acridine orange staining (n = 20). (D) TRAP+ staining to detect OC survival rate at day 8 (n = 10) (blue dotted lines indicate impressions of OCs that were present earlier). (E) Actin ring formation analyzed by phalloidin staining (n = 10). (F) qRT-PCR analyses of OC-specific genes in LysM and IFT80d/d samples (n = 3). Results are expressed as mean ± SD. Data were analyzed either using unpaired two-tailed t test or one- or two-way ANOVA as appropriate followed by Bonferroni post hoc test. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, nonsignificant.
Fig. 3.
Fig. 3.
Ctsk-Cre–mediated IFT80 deletion increases OC differentiation and bone loss. (A) Representative Western blot image demonstrating protein expression of IFT80 (80 kDa) in OCs. (B) μCT analysis of the femurs from 12-wk-old control and IFT80d/d mice (n = 5). (Scale bar, 50 µm.) Quantitative analysis of the percentage of BV/TV, Tb.Th, Tb.N, and Tb.Sp in the femurs of 12-wk-old control and cKO mice (n = 5). (C) Histological sections of tibiae from 12-wk-old control and IFT80d/d mice stained for TRAP activity (red) (n = 5). (C, Right) Images represent enlarged views. Blue arrows represent OCs derived from the blue dotted boxes (Left). Oc.S/B.S. (n = 5). (D) Number of TRAP+ stained OCs or nuclei number in OCs (n = 6). (E) Increased acidification as seen by acridine orange staining (n = 20). (F) Actin ring formation analyzed by phalloidin staining (n = 10). Results are expressed as mean ± SD. Data were analyzed using unpaired two-tailed t test. **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 4.
Fig. 4.
IFT80 associates with TRAF6 and Cbl-b to promote TRAF6 degradation. (A) Protein expression of TRAF6 (molecular mass 60 kDa), Cbl-b (120 kDa), c-Cbl (105 kDa), IFT80 (80 kDa), and β-actin (43 kDa) in LysM or IFT80d/d BMMs with or without adenoviral overexpression of IFT80 (n = 3). Samples were stimulated with RANKL for 48 h and protein changes were analyzed by Western blot. OE in superscript denotes overexpression. Graphs represent quantification of the blots. (B) Co-IP of IFT80 with TRAF6 in LysM or IFT80d/d BMMs (n = 3). (C) Co-IP of TRAF6 or IFT80 with Cbl-b in LysM or IFT80d/d BMMs (n = 3). (D) Co-IP of c-Cbl with IFT80 in LysM or IFT80d/d BMMs (n = 3). (E) Co-IP of c-Cbl with TRAF6 in LysM or IFT80d/d BMMs (n = 3). (F) Co-IP of Cbl-b with TRAF6 in LysM or IFT80d/d BMMs (n = 3). (G) Co-IP of TRAF6 with Ub (ubiquitin) in LysM, IFT80d/d, or 80OE BMMs. (H) Co-IP of Cbl-b with Ub in IFT80d/d, 80OE, or LysM BMMs. (I) Co-IP of c-Cbl with Ub in IFT80d/d, 80OE, or LysM BMMs. Immunoglobulin G (IgG) was used as a nonspecific control. For the Co-IP experiments, cells were treated with RANKL for 48 h before analysis. IB, immunoblotting. Results are expressed as mean ± SD. Data were analyzed using two-way ANOVA followed by Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, nonsignificant.
Fig. 5.
Fig. 5.
IFT80 deletion hyperactivates the RANK/RANKL and downstream AKT/ERK/GSK signaling pathways. (A) Time-course analysis and quantification of RANKL-induced pathways by Western blot in LysM or IFT80d/d BMMs (n = 3). Graphs represent quantification of the blots. (B) TRAP staining and quantification of LysM and IFT80d/d OCs treated with MK2206-Hcl (Mk), an AKT inhibitor (0.5 µM); LY294002 (Ly), a PI3K inhibitor (1 µM); and Chir99021 (Chir), a GSK3β inhibitor (1 µM) (n = 10). (C) Western blot to analyze NFATc1 expression in nuclear and cytoplasmic fractions (n = 3). Graphs represent quantification of the blots. (D) Subcellular localization of NFATc1 was analyzed by immunofluorescence. Red, nuclei; green, NFATc1 (pseudocolored); yellow denotes overlap of red and green. Results are expressed as mean ± SD. Data were analyzed using one- or two-way ANOVA as appropriate followed by Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, nonsignificant (molecular mass: pAKT/AKT: 62 kDa; pERK/ERK: 42 kDa, 44 kDa; GSK3β: 47 kDa; pP38/P38: 38 kDa; pP65/P65: 65 kDa; NFATc1: 110 kDa; LaminB1: 68 kDa; β-actin: 43 kDa).
Fig. 6.
Fig. 6.
Overexpression of IFT80 inhibits RANKL-induced bone lysis. (A) TRAP+ stained OCs and quantitative analysis in the mock and IFT80OE groups (n = 3). (B) Time-course analysis and quantification of RANKL-induced pathways by Western blot in LysM or IFT80d/d BMMs with or without IFT80 overexpression (n = 3). Graphs represent quantification of the blots. **P < 0.01, ****P < 0.0001 (molecular mass: pAKT/AKT: 62 kDa; pERK/ERK: 42 kDa, 44 kDa; GSK3β: 47 kDa; pP38/P38: 38 kDa; pP65/P65: 65 kDa; NFATc1: 140 kDa; β-actin: 43 kDa). (C) µCT analysis of the calvariae in mock or IFT80OE mice treated with RANKL (n = 5). Quantitative analysis of the percentage of BV/TV. Results are expressed as mean ± SD. Data were analyzed using one-way ANOVA followed by Bonferroni post hoc test (n = 10). ####P < 0.0001 vs. Ad-Null + RANKL; **P < 0.01, ****P < 0.0001 vs. control. (D) Schematic of the proposed mechanisms. RANKL triggers a cascade of signaling events. TRAF6 mediates the signaling pathways induced by RANKL. Under normal conditions, IFT80 bridges a link between Cbl-b and TRAF6. Cbl-b ubiquitinates TRAF6 for degradation. However, deletion of IFT80 (KO) impairs the linkage between Cbl-b and TRAF6. This likely creates a negative feedback loop, thereby promoting the autoubiquitination and degradation of Cbl-b. Absence of Cbl-b leaves TRAF6 activity unchecked, which leads to increased activation of RANK/RANKL pathways, OC numbers, and activity.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Boyle W. J., Simonet W. S., Lacey D. L., Osteoclast differentiation and activation. Nature 423, 337–342 (2003). - PubMed
    1. Teitelbaum S. L., Bone resorption by osteoclasts. Science 289, 1504–1508 (2000). - PubMed
    1. Henriksen K., Bollerslev J., Everts V., Karsdal M. A., Osteoclast activity and subtypes as a function of physiology and pathology—Implications for future treatments of osteoporosis. Endocr. Rev. 32, 31–63 (2011). - PubMed
    1. Bi H., et al. , Key triggers of osteoclast-related diseases and available strategies for targeted therapies: A review. Front. Med. (Lausanne) 4, 234 (2017). - PMC - PubMed
    1. Pedersen L. B., Rosenbaum J. L., Intraflagellar transport (IFT) role in ciliary assembly, resorption and signalling. Curr. Top. Dev. Biol. 85, 23–61 (2008). - PubMed

Publication types

-