Knockout of TLR4 promotes fracture healing by activating Wnt/β-catenin signaling pathway

Abstract

Background: The aim of this study was to investigate the effect of Toll like receptor 4 (TLR4) on fracture healing. Methods: The open tibial fracture models in TLR4 knockout (TLR4-/-) and wild type (WT) C57BL-6J mice were established. The radiological examination, tartrate-resistant acid phosphatase (TRAP) staining , Micro-CT scan and biological torsion test were performed on 7, 14 and 21 days after operation. Enzyme Linked Immunosorbent Assay (ELISA) kit was used to detect the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β) and interleukin 6 (IL-6). Western blotting was used to detect the expression of β-catenin, Wingless-type MMTV integration site family, member 4 and 5B (Wnt4 and Wnt5B), proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-2 (BMP-2) of the callus tissue obtained from mice. Results: TLR4 knockout promoted fracture healing, reduced the number of osteoclasts, increased bone callus volume (BV) and callus mineralized volume fraction (BV/TV%) (P < 0.05), increased the maximum torque and torsional stiffness of callus (P < 0.05), reduced TNF-α, IL-1β and IL-6 expression (P < 0.01), and increased the expression of β-catenin, Wnt4, Wnt5B, PCNA and BMP-2 (P < 0.01). Conclusions: TLR4 knockout reduced inflammatory and promoted fracture healing by activating Wnt/β-catenin signaling pathway.

WNT5B in Physiology and Disease

“…Indirect suppression of WNT5B and β-catenin via miRNAs that target parathyroid hormone ( Yao et al, 2018 ) and PANX3 ( Jia and Zhou, 2018 ) results in inhibition of fracture healing. Moreover, knockout of Toll-like receptor 4 (TLR4), which inhibits the WNT/β-catenin signaling pathway, promotes fracture healing and increases β-catenin, WNT4, WNT5B, PCNA, and BMP-2 ( Zhao et al, 2020 ), suggesting that β-catenin and WNT5B play similar roles in bone fracture healing. As both WNT/β-catenin-dependent and independent signaling pathways are involved in specific generative patterns of bone repair ( Heilmann et al, 2013 ; Houschyar et al, 2018 ), WNT5B might regulate β-catenin activities to generate new bone.…”

Signature of circulating small non-coding RNAs during early fracture healing in mice

“…Further, His-GTG-5′ (tiRNA-His-GTG-001) was shown to be elevated in intestinal biopsy samples of patients with an irritable bowel syndrome with diarrhea, targeting GABBR2 , TLR4 , and GABARAP which are related to, e.g., amino acid (such as glutamate) metabolism, GABAergic synapse, TNF-α signalling pathway, and insulin resistance ( Chai et al, 2021 ). Knockout of TLR4 in mice has been shown to activate Wnt/β-catenin signalling pathway, thereby promoting fracture healing ( Zhao et al, 2020 ). These data suggest that His-GTG-5′ has a role in fracture healing via regulation of macromolecular metabolism.…”

Influence of the TGF-β Superfamily on Osteoclasts/Osteoblasts Balance in Physiological and Pathological Bone Conditions

“…The canonical Wnt pathway through the β-catenin is involved in the skeletal development, as well as the fracture healing process [286,287]. For example, Wnt can direct the fate of mesenchymal stromal cells and favors their osteogenic differentiation by upregulating the genes encoding for the transcription factor Runx2 and Osterix, while it limits their adipogenic differentiation by preventing the expression of the genes encoding CCAAT/enhancer-binding protein alpha and PPAR-γ [288].…”