GIT1 contributes to autophagy in osteoclast through disruption of the binding of Beclin1 and Bcl2 under starvation condition

Abstract

Approximately 10-15% of all bone fractures do not heal properly, causing patient morbidity and additional medical care expenses. Therefore, better mechanism-based fracture repair approaches are needed. In this study, a reduced number of osteoclasts (OCs) and autophagosomes/autolysosomes in OC can be observed in GPCR kinase 2-interacting protein 1 (GIT1) knockout (KO) mice on days 21 and 28 post-fracture, compared with GIT1 wild-type (GIT1 WT) mice. Furthermore, in vitro experiments revealed that GIT1 contributes to OC autophagy under starvation conditions. Mechanistically, GIT1 interacted with Beclin1 and promoted Beclin1 phosphorylation at Thr119, which induced the disruption of Beclin1 and Bcl2 binding under starvation conditions, thereby, positively regulating autophagy. Taken together, the findings suggest a previously unappreciated role of GIT1 in autophagy of OCs during fracture repair. Targeting GIT1 may be a potential therapeutic approach for bone fractures.

Fig. 1. Osteoclast (OC) number and mineralized callus volume/tissue volume (CV/TV) are both affected in GIT1 KO mice.

a Representative TRAP staining images of the femur fracture model of GIT1 WT and GIT1 KO mice on days 21 and 28 post-fracture. Scale bars = 1000 μm. b Statistical analysis of the osteoclast surface based on the TRAP staining in GIT1 WT and GIT1 KO mice on days 21 and 28 post-fracture (values are mean ± SD, *p < 0.05, ***p < 0.001, two-tailed Student's t-tests). c Representative 2D and 3D images from micro-CT scanning in the fracture region of GIT1 WT and GIT1 KO mice on 21 and 28 days post-fracture. d The statistical analysis on mineralized callus volume/tissue volume (CV/TV, %) from micro-CT scanning. Each group contained five cases (values are mean ± SD, *p < 0.05, two-tailed Student's t-tests)

Fig. 2. The number of autophagosomes/autolysosomes in osteoclasts is decreased in GIT1 KO mice.

a Representative transmission electron microscopy (TEM) images of osteoclasts from the femur fracture model of GIT1 WT and GIT1 KO mice on days 21 and 28 post-fracture. Black arrows indicate autophagosomes/autolysosomes. Scale bars = 5 μm. b The number of autophagosomes/autolysosomes in GIT1 WT and GIT1 KO mice on days 21 and 28 during fracture repair (values are means ± SD, **p < 0.01, two-tailed Student's t-tests)

Fig. 3. The role of GIT1 in autophagy of osteoclasts and HEK293T cells.

a Amino-acid starvation in Earle’s balanced salt solution (EBSS) for different time durations (0, 0.5, 1.0, and 2.0 h) progressively increased the expression of GIT1 and LC3-II in osteoclasts. b The expression of GIT1 and LC3-II were both gradually upregulated under amino-acid starvation conditions in EBSS for different time durations (0, 1.0, and 2.0 h) in HEK293T cells. c The effectiveness of siRNA1 in knocking down GIT1 expression in osteoclasts under basal and starvation conditions. si-NC was used as the control. d The efficacy of GIT1-HA in overexpression of GIT1 in HEK293T cells under non-starvation and starvation conditions. e, f The effect of GIT1 knockdown in lowering the LC3-II level under non-starvation or starvation conditions (1 h) with or without bafilomycin A1 (Baf, 10 nM) in osteoclasts. Bafilomycin A1 was used to inhibit LC3-II degradation. Representative immunoblot images (e) and data summary (f) are shown (*p < 0.05, **p < 0.01, ns indicates no significance, Kruskal–Wallis test). g, h The effect of GIT1 overexpression in increasing the LC3-II level under basal or starvation conditions with or without bafilomycin A1 (Baf, 10 nM) in HEK293T cells. Representative images (g) and data summary (h) are shown (**p < 0.01, Kruskal–Wallis test)

Fig. 4. Knockdown of GIT1 inhibited the starvation-induced autophagic flux in OCs in vitro.

a Osteoclasts were transfected with a tandem reporter monomeric red fluorescent protein (mRFP)–green fluorescent protein (GFP)–LC3. The control and GIT1 knockdown osteoclasts with the different treatments were kept in full medium, or under starvation conditions (EBSS, 1 h). Shown are LC3 fluorescent signals from representative single cells and the cell nuclei were stained with DAPI (blue). Scale bar = 20 μm. b The number of autophagosomes/autolysosomes of osteoclasts in GIT1 knockdown and control groups under basal and starvation conditions was analyzed via TEM. Representative images of autophagosomes/autolysosomes are shown. Black arrows indicate autophagosomes and/or autolysosomes. Scale bar = 5 μm

Fig. 5. Overexpression of GIT1 promoted autophagic flux in HEK293T cells in vitro.

a, b The control and GIT1-overexpressing HEK293T cells were transfected with mRFP–GFP–LC3. Effect of GIT1 overexpression in promoting LC3 puncta formation under non-starvation and starvation conditions. Shown are LC3 fluorescent signals from representative single cells. Representative images (a) and data summary (b) are shown (**p < 0.01, Kruskal–Wallis test). Scale bar = 20 μm

Fig. 6. GIT1-regulated Beclin1 phosphorylation at the Thr119 residue.

a Representative immunoblot images showing the effect of GIT1 knockdown on the expression of p-mTOR/mTOR, p-ULK1/ULK1, p-Beclin1-S15/Beclin1, and p-Beclin1-T119/Beclin1 in the OCs under basal and starvation conditions. b Representative immunoblot images showing the effect of GIT1 overexpression on the expression of p-mTOR/mTOR, p-ULK1/ULK1, p-Beclin1-S15/Beclin1, and p-Beclin1-T119/Beclin1 in HEK293T cells under basal and starvation conditions. c-f Densitometric analysis showed the relative amounts of p-mTOR/mTOR (c), p-ULK1/ULK1 (d), p-Beclin1-S15/Beclin1 (e), and p-Beclin1-T119/Beclin1 (f) in GIT1 knockdown or GIT1-overexpressing cells compared with the relative controls under basal and starvation conditions (**p < 0.01, ns indicates no significance, Kruskal–Wallis test)

Fig. 7. GIT1 physically interacts with Beclin1 and contributes to the disruption of Beclin1-Bcl2 binding in HEK293T cells.

a–d Co-IP assays between GIT1 and Beclin1 under basal and starvation conditions. The pulling antibodies (anti-GIT1 antibody in a and anti-Beclin1 antibody in c) and the blotting antibodies are indicated. The cell lysates are displayed as input, and IgG is used as an internal control (values are means ± SD, **p < 0.01, two-tailed Student's t-tests). e, f Co-IP assays between Beclin1 and Bcl2 with or without GIT1 under basal and starvation conditions. The pulling antibodies (anti-Beclin1 antibody) and the blotting antibodies are indicated. The cell lysates are displayed as input, and IgG is used as an internal control (values are means ± SD, **p < 0.01, two-tailed Student's t-tests)