Yixuan Li1, Jingxuan Wang1,2, Fazheng Ren1,2, Wei Zhang2, Hao Zhang1, Liang Zhao1, Ming Zhang2, Wei Cui1,3, Xiaobin Wang4, Huiyuan Guo1,2. 1. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China. 2. Key Laboratory of Functional Dairy, China Agricultural University, Beijing, China. 3. Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, London, United Kingdom. 4. Department of Population, Family, and Reproductive Health, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD.
Abstract
Background: Lactoferrin (LF), as a major functional protein in dairy products, is known to modulate bone anabolic effects. However, the underlying molecular mechanisms remain unclear; the receptor of LF in osteoblast differentiation has not been identified. Objective: The aims of the study were to 1) illuminate whether the receptor of LF in osteoblast differentiation is transforming growth factor-β (TGF-β) receptor (TβR) II and 2) determine whether the TGF-β signaling pathway is activated by LF in promoting osteogenesis in vitro and in vivo, in addition to P38 and extracellular signal-regulated kinase (ERK) pathways. Methods: We utilized co-immunoprecipitation to detect any binding of LF to TβRII. Subsequently, the role of the TGF-β signaling pathway involved in LF-induced osteoblast proliferation and differentiation was determined by inhibition of TβRI activity by inhibition and knockout of TβRII expression by small guide RNA (sgRNAs) in MC3T3-E1 cells. In addition, 4-wk-old male C57BL/6J mice were orally administered 100 mg LF/kg body weight for 16 wk, after which any activation of the TGF-β signaling pathway in vivo was measured by Western blots. Results: LF was shown to directly interact with the TβRII protein and to activate the TGF-β signaling pathway in MC3T3-E1 cells. Inhibition of TβRI activity and knockout TβRII expression both attenuated the stimulation of LF in osteoblast proliferation and differentiation by 30-50%. LF-induced activation of TGF-β canonical signaling resulted in upregulation of osteogenic factors. Moreover, the expression of phosphorylated-drosophila mothers against decapentaplegic protein 2 (SMAD2) was increased by 1-fold after LF treatment in the femoral tissue of mice. Conclusions: This study provides evidence identifying TβRII as an LF receptor in LF-induced osteoblast differentiation. In addition, the TβRII-dependent TGF-β canonical signaling pathways were proven to play an important role in mediating LF-induced osteogenesis both in MC3T3-E1 cells and in C57BL/6J mice.
Background: Lactoferrin (LF), as a major functional protein in dairy products, is known to modulate bone anabolic effects. However, the underlying molecular mechanisms remain unclear; the receptor of LF in osteoblast differentiation has not been identified. Objective: The aims of the study were to 1) illuminate whether the receptor of LF in osteoblast differentiation is transforming growth factor-β (TGF-β) receptor (TβR) II and 2) determine whether the TGF-β signaling pathway is activated by LF in promoting osteogenesis in vitro and in vivo, in addition to P38 and extracellular signal-regulated kinase (ERK) pathways. Methods: We utilized co-immunoprecipitation to detect any binding of LF to TβRII. Subsequently, the role of the TGF-β signaling pathway involved in LF-induced osteoblast proliferation and differentiation was determined by inhibition of TβRI activity by inhibition and knockout of TβRII expression by small guide RNA (sgRNAs) in MC3T3-E1 cells. In addition, 4-wk-old male C57BL/6J mice were orally administered 100 mg LF/kg body weight for 16 wk, after which any activation of the TGF-β signaling pathway in vivo was measured by Western blots. Results:LF was shown to directly interact with the TβRII protein and to activate the TGF-β signaling pathway in MC3T3-E1 cells. Inhibition of TβRI activity and knockout TβRII expression both attenuated the stimulation of LF in osteoblast proliferation and differentiation by 30-50%. LF-induced activation of TGF-β canonical signaling resulted in upregulation of osteogenic factors. Moreover, the expression of phosphorylated-drosophila mothers against decapentaplegic protein 2 (SMAD2) was increased by 1-fold after LF treatment in the femoral tissue of mice. Conclusions: This study provides evidence identifying TβRII as an LF receptor in LF-induced osteoblast differentiation. In addition, the TβRII-dependent TGF-β canonical signaling pathways were proven to play an important role in mediating LF-induced osteogenesis both in MC3T3-E1 cells and in C57BL/6J mice.
Authors: Yan Xu; Jing-Jing An; Dina Tabys; Yin-Dan Xie; Tian-Yu Zhao; Hao-Wei Ren; Ning Liu Journal: Int J Mol Sci Date: 2019-09-28 Impact factor: 5.923