Increased Expression of FGF-21 Negatively Affects Bone Homeostasis in Dystrophin/Utrophin Double Knockout Mice

Abstract

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy seen in children. In addition to skeletal muscle, DMD also has a significant impact on bone. The pathogenesis of bone abnormalities in DMD is still unknown. Recently, we have identified a novel bone‐regulating cytokine, fibroblast growth factor‐21 (FGF‐21), which is dramatically upregulated in skeletal muscles from DMD animal models. We hypothesize that muscle‐derived FGF‐21 negatively affects bone homeostasis in DMD. Dystrophin/utrophin double‐knockout (dKO) mice were used in this study. We found that the levels of circulating FGF‐21 were significantly higher in dKO mice than in age‐matched WT controls. Further tests on FGF‐21 expressing tissues revealed that both FGF‐21 mRNA and protein expression were dramatically upregulated in dystrophic skeletal muscles, whereas FGF‐21 mRNA expression was downregulated in liver and white adipose tissue (WAT) compared to WT controls. Neutralization of circulating FGF‐21 by i.p. injection of anti‐FGF‐21 antibody significantly alleviated progressive bone loss in weight‐bearing (vertebra, femur, and tibia) and non–weight bearing bones (parietal bones) in dKO mice. We also found that FGF‐21 directly promoted RANKL‐induced osteoclastogenesis from bone marrow macrophages (BMMs), as well as promoted adipogenesis while concomitantly inhibiting osteogenesis of bone marrow mesenchymal stem cells (BMMSCs). Furthermore, fibroblast growth factor receptors (FGFRs) and co‐receptor β‐klotho (KLB) were expressed in bone cells (BMM‐derived osteoclasts and BMMSCs) and bone tissues. KLB knockdown by small interfering RNAs (siRNAs) significantly inhibited the effects of FGF21 on osteoclast formation of BMMs and on adipogenic differentiation of BMMSCs, indicating that FGF‐21 may directly affect dystrophic bone via the FGFRs‐β‐klotho complex. In conclusion, this study shows that dystrophic skeletal muscles express and secrete significant levels of FGF‐21, which negatively regulates bone homeostasis and represents an important pathological factor for the development of bone abnormalities in DMD. The current study highlights the importance of muscle/bone cross‐talk via muscle‐derived factors (myokines) in the pathogenesis of bone abnormalities in DMD. © 2019 American Society for Bone and Mineral Research.

Skeletal Muscle and Bone – Emerging Targets of Fibroblast Growth Factor-21

“…Although the exact mechanism of how FGF21 regulates bone homeostasis is still not clear, several direct and indirect mechanisms have been proposed (Figure 2). The expression of FGFRs/β-klotho in bone tissue has not been fully established, however, a recent study on dystrophic mice demonstrated that β-klotho and FGFR expression is significantly induced in mature osteoclasts (Li et al, 2020). These data indicate that bone is a direct target of FGF21 and FGF21 may affect osteoclastogenesis in DMD.…”

“…Myokines are cytokines or peptides synthesized and released by muscle in response to muscular contraction or various stimuli (Pedersen et al, 2007). Under basal conditions, the expression of FGF21 is predominantly from liver and adipose tissue (Nishimura et al, 2000), however, the expression and secretion of FGF21 from skeletal muscle is significantly increased under certain conditions, such as mitochondrial dysfunction (Keipert et al, 2014;Lehtonen et al, 2016;Steele et al, 2016;Khan et al, 2017;Romanello et al, 2019), muscular dystrophy (Lovadi et al, 2017;Zhou et al, 2018;Li et al, 2020), and exercise (Ost et al, 2016;Kruse et al, 2017;Morville et al, 2018). Thus, in addition to being a hepatokine and adipokine, FGF21 is also well-established as a myokine (Pedersen and Febbraio, 2012;Itoh, 2014;Pereira et al, 2017).…”

“…Elevated serum FGF21 has been demonstrated in animal models of Duchenne muscular dystrophy (DMD; Zhou et al, 2018;Li et al, 2020) and is primarily derived from dystrophic muscle (Li et al, 2020). However, the mechanisms that drive the expression of FGF21 from dystrophic skeletal muscle is still largely unknown.…”

“…However, the mechanisms that drive the expression of FGF21 from dystrophic skeletal muscle is still largely unknown. Mitochondrial deficiency (Timpani et al, 2015), autophage dysfunction (De Palma et al, 2012), and endoplasmic reticulum (ER) stressors (Pauly et al, 2017) have been implicated as part of the pathogenesis of DMD and have also been shown to increase the expression of FGF21 from skeletal muscle (Zhou et al, 2018;Li et al, 2020). Furthermore, one of the hallmarks of DMD pathology is constant muscle degeneration and regeneration (Rosenberg et al, 2015).…”

“…Increased serum FGF21 has also been reported to negatively affect bone mineral density (BMD) during lactation in C57BL/6 mice (Bornstein et al, 2014). Moreover, in a mouse model of DMD, which shows elevated circulating skeletal muscle-derived FGF21 (Zhou et al, 2018), blockage of FGF21's action using a neutralization antibody, resulted in significantly increased bone mass and improved quality of bone tissues (Li et al, 2020). However, conflicting data exists as others have reported no bone loss observed in FGF21 KO mice nor in recombinant FGF21 treated mice (Li et al, 2017).…”