TRPA1 aggravates osteoclastogenesis and osteoporosis through activating endoplasmic reticulum stress mediated by SRXN1

Abstract

Osteoporosis (OP) is a disorder of bone remodeling caused by an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, inhibiting excessive osteoclast activity is one of the promising strategies for treating OP. A major transient receptor potential cation channel, known as transient receptor potential ankyrin 1 (TRPA1), was found to alleviate joint pain and cartilage degeneration in osteoarthritis. However, little research has focused on TRPA1 function in OP. As a result, this study aimed to explore the TRPA1 characteristics and its potential therapeutic function during osteoclastogenesis. The TRPA1 expression gradually increased in the osteoclast differentiation process; however, its suppression with small interfering RNA and an inhibitor (HC030031) significantly controlled the osteoclast count and the expression of osteoclast characteristic genes. Its suppression also inhibited endoplasmic reticulum (ER) stress-related pancreatic ER kinase (PERK) pathways. An ER stress inhibitor (thapsigargin) reversed the down-regulated levels of ER stress and osteoclast differentiation by suppressing TRPA1. Transcriptome sequencing results demonstrated that TRPA1 negatively regulated reactive oxygen species (ROS) and significantly increased the expression of an antioxidant gene, SRXN1. The osteoclast differentiation and the levels of ER stress were enhanced with SRXN1 inhibition. Finally, TRPA1 knockdown targeting macrophages by adeno-associated virus-9 could relieve osteoclast differentiation and osteopenia in ovariectomized mice. In summary, silencing TRPA1 restrained osteoclast differentiation through ROS-mediated down-regulation of ER stress via inhibiting PERK pathways. The study also indicated that TRPA1 might become a prospective treatment target for OP.

Fig. 1. The establishment of the OVX-induced OP mouse model and TRPA1 was upregulated after surgery and RANKL intervention.

A Representative pictures revealing that the femur osteopenia was induced by OVX. B Quantitative analysis of bone microstructure and cortical bone parameters. C, D Immunofluorescence staining and quantitation of NFATc1 and TRPA1 after surgery. N = 7 per group. E, F Immunofluorescence staining and quantitation of TRPA1 under RANKL intervention. Student’s t-test was applied to determine statistical significance. G, H Western blot and quantitation for TRPA1 expression in a dose-dependent manner for proteins of TRPA1 in a dose-dependent manner. I Intracellular calcium ion level in a dose-dependent manner. N = 3 per group. One-way ANOVA with Tukey’s multiple-comparison test was conducted to determine statistical significance (*p < 0.05, **p < 0.01).

Fig. 2. TRPA1 inhibition suppressed osteoclast differentiation and function.

A, B Representative pictures of TRAcP staining and quantification of the TRAcP-positive cells (nuclei >3) under Si-TRPA1 intervention. C, D Representative pictures of bone resorption assay and quantification of resorbed area under Si-TRPA1 intervention. E, F Immunofluorescence staining and quantitation of TRPA1 and NFATc1 under Si-TRPA1 intervention. G Western blot and quantitation for proteins of TRPA1, MMP9, and NFATc1 under Si-TRPA1 intervention. H The relative mRNA expression of TRPA1, MMP9, and NFATc1 under Si-TRPA1 intervention. I Intracellular calcium ion level under Si-TRPA1 intervention. N = 3 per group. One-way ANOVA with Tukey’s multiple-comparison test was conducted to determine statistical significance (**p < 0.01).

Fig. 3. TRPA1 inhibition suppressed osteoclast differentiation and function by alleviating ER stress.

A Western blot and quantitation for proteins of PERK/p-PERK, eIF2α/p- eIF2α, ATF4, and CHOP under Si-TRPA1 intervention. B Western blot and quantitation for proteins of TRPA1, MMP9, and NFATc1 under Si-TRPA1 and thapsigargin intervention. C, D Representative pictures of TRAcP staining and quantification of the TRAcP-positive cells (nuclei >3) under Si-TRPA1 and thapsigargin intervention. E, F Representative pictures of bone resorption assay and quantification of resorbed area under Si-TRPA1 and thapsigargin intervention. G, H Representative images and quantification of irregular ER intervened by Si-TRPA1 and thapsigargin under TEM. Red arrows represented ER. I, J Immunofluorescence staining of calnexin‐ER marker and quantification of ER pyknosis under Si-TRPA1 and thapsigargin intervention. N = 3 per group. One-way ANOVA with Tukey’s multiple-comparison test was conducted to determine statistical significance (*p < 0.05, **p < 0.01).

Fig. 4. TRPA1 inhibition suppressed osteoclast differentiation via alleviating ER stress mediated by SRXN1.

A The differentially expressed mRNAs in RAW 264.7 cells in response to Si-TRPA1 are illustrated as a heatmap. B GO enrichment of differential genes. C Heatmap of differential genes in negative regulation of reactive oxygen species biosynthetic process. D The relative mRNA expression of MMP9 and NFATc1 under Si-TRPA1 and Si-SRXN1 intervention. E Western blot and quantitation for proteins of MMP9 and NFATc1 under Si-TRPA1 and Si-SRXN1 intervention. F, G Representative images of TRAcP staining and quantification of the TRAcP-positive multinucleated cells (nuclei >3) under Si-TRPA1 and Si-SRXN1 intervention. H, I Representative images of bone resorption assay and quantification of the resorbed area under Si-TRPA1 and Si-SRXN1 intervention. N = 3 per group. One-way ANOVA with Tukey’s multiple-comparison test was conducted to determine statistical significance (*p < 0.05, **p < 0.01).

Fig. 5. TRPA1 inhibition suppressed osteoclast differentiation via alleviating ER stress mediated by SRXN1.

A, B Flow cytometric analysis and quantitation of ROS-positive cells under Si-TRPA1 and Si-SRXN1 intervention. C, D Representative images of and quantitation of ROS-positive cells under Si-TRPA1 and Si-SRXN1 intervention. E, F Representative images and quantification of irregular ER intervened by Si-TRPA1 and Si-SRXN1 and thapsigargin under TEM. Red arrows represented ER. G, H Immunofluorescence staining of calnexin‐ER marker and quantification of ER pyknosis under Si-TRPA1 and Si-SRXN1 intervention. I Western blot and quantitation for proteins of PERK/p-PERK, eIF2α/p- eIF2α, ATF4, and CHOP under Si-TRPA1 and Si-SRXN1 intervention. N = 3 per group. One-way ANOVA with Tukey’s multiple-comparison test was conducted to determine statistical significance (*p < 0.05, **p < 0.01).

Fig. 6. TRPA1 inhibition prevents OVX-induced bone loss in vivo.

A, B Immunofluorescence staining and quantitation of TRPA1 under AAV9-TRPA1 intervention. C Representative pictures revealed that the femur bone loss was alleviated under AAV9-TRPA1 intervention. D Quantitative analysis of parameters regarding bone microstructure and cortical bone. E, F H&E staining and quantitative analysis of histomorphometric bone parameters of BV/TV (%). G, H TRAcP staining and quantitative analysis of TRAcP+ osteoclast number in the bone sections. I, J Immunofluorescence staining and quantitation of NFATc1 and CHOP under AAV9-TRPA1 intervention. N = 7 per group. One-way ANOVA with Tukey’s multiple-comparison test was conducted to determine statistical significance (**p < 0.01).