Rc3h1 negatively regulates osteoclastogenesis by limiting energy metabolism

Abstract

Rationale: Osteoclasts are giant bone-resorbing cells that need vigorous mitochondrial respiration to support their activation. Rc3h1, an RNA-binding protein, precisely governs the homeostasis of mRNA. However, the precise role of Rc3h1 in regulating iron metabolism and mitochondrial respiration in osteoclasts is not yet understood. Methods: We generated Rc3h1-deficient mice in osteoclast precursors and mature osteoclasts. The bone mass and osteoclast activity in bone tissues were evaluated. Moreover, we assessed the differentiation, bone resorption, iron content, and mitochondrial function of osteoclasts in vitro. In the end, the target gene of Rc3h1 and its role in mediating the effect of Rc3h1 on mitochondrial respiration in osteoclasts were further investigated. Results: Mice lacking Rc3h1 exhibit low bone mass. In addition, Rc3h1 deletion in osteoclasts significantly promotes osteoclast activation. Mechanistically, Rc3h1 post-transcriptionally represses the expression of transferrin receptor 1 (Tfr1), restricting iron absorption and mitochondrial respiration in osteoclasts. Inhibition of Tfr1 in Rc3h1-deficient osteoclasts diminishes excessive osteoclast formation and mitochondrial respiration. Conclusion: These findings suggest that Rc3h1 has a negative effect on osteoclast activation via limiting iron resorption and mitochondrial respiration. Finally, targeting the Rc3h1/Tfr1 axis might represent a potential therapeutic approach for bone-loss diseases.

Keywords: Rc3h1; Tfr1; mitochondria; osteoclast; osteoporosis.

Figure 1

Low bone mass was exhibited in Rc3h1^LysM mice. (A) Protein level of Rc3h1 during osteoclast formation induced by M-CSF and RANKL from C57BL/6J mice. (B) The protein expression of Rc3h1 in Rc3h1^flox and Rc3h1^LysM osteoclasts. (C-D) Representative micro-CT images and quantitative analysis of proximal tibia trabecular and cortical bone in 12-week-old male Rc3h1^flox (n = 8) and Rc3h1^LysM mice (n = 11). (E-F) Representative micro-CT images and quantitative analysis of proximal tibia trabecular and cortical bone in 12-week-old female Rc3h1^flox (n = 7) and Rc3h1^LysM (n = 7) mice. (G) TRAP staining and analysis of proximal tibia from Rc3h1^flox (n = 5) and Rc3h1^LysM (n = 5) mice. Scale bar= 200 μm. (H-I) The ELISA of CTX-1 and PINP from Rc3h1^flox (n = 8) and Rc3h1^LysM (n = 11) serum. (J) Double calcein label of proximal tibia bone in Rc3h1^flox (n = 5) and Rc3h1^LysM (n = 5) mice. Scale bar= 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Deletion of Rc3h1 in differentiated osteoclasts leads to bone loss. (A-B) Representative 3D micro-CT reconstruction images and quantitative analysis of distal femur trabecular and cortical bone in 12-week-old male Rc3h1^flox (n = 12) and Rc3h1^Ctsk (n = 12) mice. (C-D) Micro-CT scanning and analysis of distal femur trabecular and cortical bone in 12-week-old female Rc3h1^flox (n = 6) and Rc3h1^Ctsk (n = 6) mice. (E-F) TRAP staining was used to quantitatively analyze the number of osteoclasts in distal femur bone tissue in 12-week-old male Rc3h1^flox (n = 6) and Rc3h1^Ctsk (n = 6) mice. (G) The serum CTX-1 was measured by ELISA. Scale bar= 200 μm (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Loss of Rc3h1 accelerates osteoclast formation. (A) In vitro TRAP staining was performed on osteoclasts from Rc3h1^flox and Rc3h1^LysM mice. Scale bar= 200 μm. (B) Podosome staining on Rc3h1^flox and Rc3h1^LysM osteoclasts. Scale bar= 200 μm. (C) Representative images of acidification in osteoclasts from Rc3h1^flox and Rc3h1^LysM osteoclasts. Scale bar= 300 μm. (D) Representative images of scanning electron microscopy on bone resorption pits from Rc3h1^flox and Rc3h1^LysM osteoclasts. Scale bar= 100 μm. (E) Expression levels of osteoclast-related genes on BMMs with the induction of M-CSF and RANKL for 3 days assessed using qPCR. (F-I) Proteins and phosphorylation states of ERK, P38, and JNK upon the stimulation of RANKL. (J-K) The WB of Rc3h1 and TRAP staining of osteoclasts stimulated with RANKL and M-CSF for 6 days from BMMs transfected with Lv-vector or Lv-Rc3h1. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Tfr1 is a candidate target of Rc3h1 in osteoclasts. (A-B) Volcano plot and heatmap of differentially expressed genes in Rc3h1^flox and Rc3h1^LysM osteoclasts induced by M-CSF and RANKL for 3 days (n = 3). (C) Heatmap of iron metabolism-related genes in Rc3h1^flox and Rc3h1^LysM osteoclasts. (D-E) Immunofluorescent staining and quantitative analysis of Tfr1 on proximal tibia bone slides from Rc3h1^flox and Rc3h1^LysM mice (n = 4). Scale bar= 250 μm. (F) mRNA level of Tfr1 using qPCR in WT and Rc3h1-deficient osteoclasts. (G-H) Protein level of Tfr1 during the formation of osteoclasts in WT and Rc3h1-deficient osteoclasts. (I-J) Results of mean fluorescent intensity of FerroOrange from WT and Rc3h1-deficient osteoclasts using flow cytometry to detect cellular Fe²⁺content. (K) Total iron from lysed otseoclasts measured by absorbance at 593 nm. (L-M) Western bolts were presented to confirm the Rc3h1-IP efficiency, and qPCR was used to quantify the indicated mRNA in the IP and IgG groups. RIP was performed on RAW264.7 cells transfected with the Rc3h1-Puro-Flag vector after RANKL stimulation for 3 days (n= 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Loss of Rc3h1 in osteoclasts results in enhanced mitochondrial respiration. (A-C) Flow cytometry analysis of osteoclasts' mitochondrial mass, ROS, and membrane potential stained with mitogreen, mitoSOX, and TMRM, respectively. (D) Relative cellular ATP content in WT and Rc3h1-deficient osteoclasts. (E) Western blot evaluation of mitochondrial respiration complexes C-I to C-III, C-V and CTSK in Rc3h1^flox and Rc3h1^LysM osteoclasts. (F-G) Extracellular oxygen consumption rate (OCR) analysis and statistical analysis of Rc3h1^flox and Rc3h1^LysM osteoclasts using seahorse assay. All the osteoclasts were derived from Rc3h1^flox and Rc3h1^LysM BMMs after M-CSF and RANKL induction for 3 days. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Tfr1 mediates the effects of Rc3h1 on osteoclastogenesis and mitochondrial respiration. (A-B) Representative TRAP staining images and quantitative analysis of osteoclasts induced by M-CSF and RANKL 5 days with the presence of siTfr1 or Ferristatin II. Scale bar= 200 μm. (C) OCR analysis of Rc3h1^flox and Rc3h1^LysM osteoclasts using seahorse assay with or without the presence of siTfr1. (D) The mRNA expression of Tfr1 in osteoclasts treated with negative control or siTfr1 using qPCR. (E) The protein expression of Tfr1 in osteoclasts treated with negative control or siTfr1. (F) The diagram of the mechanism of Rc3h1 regulating mitochondrial respiration in osteoclasts (created in BioRender.com). *P < 0.05, **P < 0.01, ***P < 0.001.