The Effect And Its Mechanism Of FGFR1 On The Differentiation And Activation Of Mouse Osteoclasts

Osteoclast, derived from bone marrow monocytes, is a specific kind of mature macrophages in bone tissue. Multiple molecules, such as cytokines, hormones and transcription factors, can regulate the differentiation and activation of osteoclasts indirectly by affecting the expression of nuclear factor NF-кB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colonystimulating factor (M-CSF) in osteoblast. In addition, estrogens and fibroblast growth factors (FGFs) can also regulate the differentiation and activation of osteoclasts directly by binding to their receptors in the osteoclasts.FGFs are important moleculars regulating the proliferation and differentiation of cells. Data indicated that FGFs, such as FGF2, FGF9, FGF18 and FGF23 can regulate the differentiation and activation of osteoclasts. Since FGFs exert their biological effects through their high affinity receptors, fibroblast growth factor receptors (FGFRs) on cell membrane, it's speculated that FGFRs can regulate the function of osteoclasts by transmiting the FGFs signal. FGFR1 is expressed in osteoclasts and can be phosphorylated in osteoclasts by FGF2. These data indicated that FGFR1 may regulate the function of osteoclast directly, but there was no direct genetic evidence. The underlying mechanisms also need to be explored. Moreover, FGFR1 is also expressed in osteoblasts, and the bone resorption function of osteoclasts is impaired in mice with conditional deficiency of Fgfr1 in immature osteoblasts. Above data indicate that FGFR1 may play an important role in the indirect regulation of osteoclasts through osteoblasts, but the molecular mechanism is not clarified. Moreover, the regulatory effect of osteoblasts on the differentiation and activation of osteoclasts is dependent on the differentiation stage of osteoblasts. In adult stage, osteoblasts in bone tissue are mainly mature osteoblasts and osteocytes. It's needed to see if FGFR1 can indirectly regulate the differentiation and activation of osteoclasts through mature osteoblasts, and its underlying molecular mechanisms is also needed to be explored,.To reveal the direct and indirect effect of FGFR1 on the differentiation and activation of osteoclasts, and the underlying molecular mechanisms, mice with Fgfr1 deficiency in either osteoclasts or mature osteoblasts were used.Main methodsPart I Exploration of the direct effect and its mechanism of FGFR1 on the differentiation and activation of osteoclasts in vivo and in vitro. Animals and groups: Mice with Fgfr1 being deleted specifically in osteoclasts (Lyzs-Cre; Fgfr1f/f) (Mutants) and their littermate controls (Fgfr1f/f) (controls)1. In Vivo assay1) Radiographic analysis and safranine O/fast green (SO/FG) staining were used to detect the changes of bone remodeling of tibiae in 4-month-old mutants and their equivalent littermate controls. The differentiation and activation of osteoclasts were evaluated by tartrate resistant acid phosphatase (TRAP) staining.2) To further investigate the effect of FGFR1 on the differentiation and activation of osteoclasts during bone regeneration, non-stablized tibiae fractures in 4-month-old mutants and littermate controls were generated. The fracture healing of these mice were observed using radiographic analysis and SO/FG staining. The differentiation and activation of osteoclasts during the fracture healing process were detected by TRAP staining.2. In Vitro assay1) Mouse bone marrow monocytes were isolated from 6-week-old mutants and littermate controls, and cultured with M-CSF and RANKL. The differentiation and bone resorption activity of osteoclasts were evaluated by quantifying the number of TRAP positive osteoclasts and the area of bone resorption pits.2) Total RNA and protein were extracted from the primary osteoclasts induced by M-CSF and RANKL for 8 days, and the expression of TRAP, Ctsk, and MMP-9 and the activation of MAPK (p38, Erk, and JNK) in osteoclasts were detected by Real-Time PCR and Western blot.Part II Exploration of the indirect effect and its mechanism of FGFR1 on the differentiation and activation of osteoclasts in vitro.Animals and groups: Mice with Fgfr1 being deleted specifically in mature osteoblasts (OC-Cre; Fgfr1f/f) (mutants) and littermate controls (Fgfr1f/f) (controls)1. Primary osteoblasts were isolated from the calvrial bone of newborn mutants and controls; the growth curve of osteoblasts was obtained by direct counting. Apoptosis of osteoblasts was detected by TUNEL assay.2. Total RNA and protein were extracted from the third passage osteoblasts, and the expression of RANKL, OPG, and M-CSF, and the activation of MAPK in osteoblasts were detected by Real-Time PCR and Western blot.3. The third passage osteoblasts from either mutants or controls were co-cultured with myelomonocytes isolated from wild type mice. The differentiation and bone resorption activity of osteoclasts were observed by checking the number of TRAP positive osteoclasts and the area of resorption pits.Main resultsPart I FGFR1 directly promotes the differentiation and activation of osteoclasts by activting the Erk MAP kinase1. There was no significant difference in the bone density, the number of trabecular bone, and TRAP-positive osteoclasts between 4-month-old mutants and littermate controls. We thus further studied the effect of FGFR1 on the differentiation and bone resorption activity of osteoclasts during bone fracture healing. X-ray analysis showed that there was no significant difference between mutants and controls at 10 days of fracture healing, however, the optical density of callus in mutants was significantly lower than that in controls at 14, 21 and 28 days after fracture healing. Furthermore, the fracture healing was delayed in mutants at day 21 after fracture in mutants when compared with equvalent controls. Histological analysis indicated that the degradation of cartilage calluses was obviously impaired as a result of the decreased number and spreading area of osteoclasts (P<0.05) on day 14 and 21. These results indicated that FGFR1 appears not to affect the bone remodeling and osteoclasts function in 4-month-old mice. However, it may promote the differentiation and activation of osteoclasts in the process of fracture healing.2. The number of osteoclasts induced from mutant bone marrow by RANKL and M-CSF and the area of resorption pits were significantly reduced (P<0.05). The spread of mutant osteoclasts on bone slice was impaired. The expression of TRAP and MMP-9 and the phosphorylation of Erk MAP kinase was also down-regulated obviously (P<0.05) in mutant osteoclasts compared with that of controls. These results indicated that FGFR1 could promote the differentiation and bone resorption activity of osteoclasts by up-regulating the expression of TRAP and MMP-9 and activating Erk MAP kinase in osteoclasts.Part II FGFR1 indirectly promotes the differentiation and activation of osteoclasts by up-regulating the expression of MCSF and down-regulating the expression of OPG in osteoblast.The proliferation and apoptosis of osteoblasts derive from mutants were enahanced significantly compared with that of controls (P<0.05). The expression of M-CSF and the phosphorylation of Erk and p38 MAP kinase in mutant osteoblasts were reduced markedly (P<0.05). Increased expression of OPG was also observed (P<0.05) in mutant osteoblasts. The number of TRAP positive osteoclasts and area of resorption pits in the co-cultured system with mutant osteoblasts were also reduced (P<0.05). These results indicated that FGFR1 can inhibit the proliferation and apoptosis of osteoblasts, and up-regulate the phosphorylation of Erk and p38 MAP kinase and the expression of M-CSF in osteoblasts. Moreover, FGFR1 can also down-regulate the expression of OPG in osteoblasts and thus indirectly promote the differentiation and bone resorption activity of osteoclasts in the co-culture system.Major conclusions1. FGFR1 appears not to influence the differentiation and activition of osteoclasts during bone remodeling in 4-month-old mice. However, FGFR1 may promote the differentiation and activation of osteoclasts during bone fracture healing.2. FGFR1 direclty promotes the osteoclastogenesis and bone resorption by up-regulating the expression of TRAP and MMP-9, and the phosphorylation of Erk MAP kinase in osteoclasts. 3. FGFR1 inhibits the proliferation and apoptosis of osteoblasts. Moreover, FGFR1 may indirectly promote the differentiation and activation of osteoclasts in co-culture systerm by up-regulating the phosphorylation of p38 and Erk MAP kinase, the expression of M-CSF in osteoblasts., and down-regulateing the expression of OPG in osteoblasts.