Palovarotene inhibits the NF‐κB signalling pathway to prevent heterotopic ossification

Abstract

Heterotopic ossification (HO) is a common disease characterized by pain, dysfunction and calcification. The mechanisms underlying HO have not been completely elucidated. Palovarotene, a retinoic acid receptor γ agonist, significantly inhibits the formation of HO in vivo. However, its specific mechanism of action remains unclear. Therefore, we aimed to evaluate the signalling pathways related to the formation of HO as well as the mechanism of palovarotene action. We constructed in vitro and in vivo models of HO. Osteogenic differentiation of bone mesenchymal stem cells (BMSCs) was observed by alizarin red and alkaline phosphatase staining assays in vitro. X‐ray and haematoxylin–eosin staining were performed in vivo. Western blots and reverse transcription‐polymerase chain reaction were performed to determine the levels of osteogenic‐ and inflammation‐related genes. Immunofluorescence and immunocytochemistry were used to assess the levels of p65, the core molecule of the nuclear factor κ‐B (NF‐κB) signalling pathway. We demonstrated that, in vitro, under inflammatory stimulation, pathological calcium deposition increased in BMSCs. The levels of osteogenesis‐ and inflammation‐related genes were also upregulated, along with an enhanced expression of p65. Immunofluorescence assays revealed that p65 entered the nucleus, thereby stimulating the downstream effectors of the NF‐κB pathway. The above trends were reversed after palovarotene treatment. In conclusion, the NF‐κB signalling pathway played an important role in HO, and palovarotene could alleviate HO by blocking the NF‐κB cascade. Our results may provide a theoretical basis for palovarotene in the treatment of HO. Further studies on the side effects of palovarotene are warranted in the future.

Role of the NF-kB signalling pathway in heterotopic ossification: biological and therapeutic significance

“…These findings suggest that different regulatory effects occur via different modulators that can affect NF-κB activity to exert different effects. In addition, in FOP, ACVR1 mutation-induced high TLR4 sensitivity can activate NF-κB signalling and interact with smad signalling in macrophages to promote inflammation in MSCs, and mTOR signalling may be involved in chondrogenic and osteogenic commitment to promote ectopic bone formation during FOP [ 16 , 100 , 102 , 126 , 128 ].…”

“…Smad signalling is important for the expression of osteogenic genes, including OCN, Sox9 and Runx2. Osteogenic gene expression can alter MSC fate by accelerating MSC differentiation into preosteoblasts for final ectopic bone formation [ 128 ]. Moreover, it has recently been demonstrated that during chondrogenesis triggered by activin A, mesenchymal stromal cells obtained from patients with FOP exhibit increased mTOR signalling [ 100 , 102 ].…”

“…For example, TSCs function as osteogenic progenitor cells in an inflammatory microenvironment dominated by polarized macrophages; in addition, TSCs undergo osteogenic differentiation, during which the levels of bone deposition and phosphatase are increased, the NF-κB and smad pathways are activated, and NF-κB is transported into the nucleus where it promotes the transcription of NF-κB-dependent genes [ 127 ]. The transcription factors of NF-κB, including Smad1/5/8, are phosphorylated by ACVR1 after activin binding, and phosphorylated Smad1/5/8 binds with Smad4 and enters the nucleus to regulate gene expression [ 128 ]. Ultimately, HO can be induced.…”

Intersections of Fibrodysplasia Ossificans Progressiva and Traumatic Heterotopic Ossification

“…Burn/tenotomy HO mouse models have also shown early increased levels of IL-6 and IL-1α in mice that form HO [ 187 ]. Recent studies have shown that the NF-κB signaling pathway plays an important role in tHO; when the NF-κB cascade was blocked, HO formation significantly decreased [ 188 ]. Similar to TGF-β pathways implicated in FOP, a recent study in a burn mouse model revealed that TGF-β1-producing macrophages are associated with HO, and a systemic reduction in macrophage-produced TGF-β levels helped to ameliorate HO [ 186 ].…”

The role of miRNA and lncRNA in heterotopic ossification pathogenesis

“…Palovarotene, a retinoic acid receptor gamma (RARγ) agonist, previously used in patients with emphysema [ 106 ], can inhibit the chondrogenesis phase in HO formation. Palovarotene influences BMP/SMAD-dependent pathway [ 107 ] and NF-κB signaling [ 108 ] preventing HO formation via endochondral ossification. In a trauma-induced HO rat model.…”