The Mechano-growth Factor E Peptide Regulates Bone Formation And Resorption In Vitro

Insulin-like growth factor I (IGFI), a single-chain polypeptide of70amino acids, isan important growth factor of bone matrix and has important roles in regulating bonemetabolism. Alternate splicing within the E domain of the IGFI gene produces threedistinct isoforms: IGFIEa, IGFIEb (in rat), and IGFIEc (in human). IGFIEb and IGFIEcare also known as mechano-growth factor (MGF) because they are often discovered inskeletal muscle or osteoblasts subjected to stretch or injury. MGF was discovered by theresearch group of Goldspink in1996. Since then many studies have confirmed thatMGF is an important local tissue repair factor. Structural characterization of MGFidentified a unique C-terminal E domain contains24amino acids (MGF-Ct24E) thatendows MGF with functions distinct from mature IGF-I and IGF-IEa. Previous studieshave demonstrated that MGF is a key factor involved in mediating mechanicalstimulation or tissue injury induced satellite cell proliferation and differentiation in vivo.Consistent with the function of MGF, MGF-Ct24E induces proliferation of myoblastsand has a strong neuroprotective effect in brain ischemia, suggesting that MGF-Ct24Eitself, once cleaved from the mature protein, has intrinsic biological activities: tissueprotection, repair and adaptation. Our previous studies even found that MGF wasinitially shown to participate in the response to injury in bone cells (or tissues) in anautocrine/paracrine manner, suggesting a potentially positive role in bone remodeling.Subsequently, MGF-Ct24E has been shown to accelerate fracture healing in a rabbitmodel by enhancing osteoblasts proliferation. The mechanisms of MGF-Ct24E functionin the regulation of bone growth, development, and injury repair are, however, far fromunderstood. Only our laboratory studies show that the MGF-Ct24E appears to stimulateproangiogenic activities in human vascular endothelial cells. According to the previousresearch results of our team on MGF-Ct24E, the studies are undertaken to investigatethe roles and mechanism of MGF-Ct24E on bone formation and absorption, in order todevelop therapies for bone repair using MGF-Ct24E. The main works and conclusionsare included as follows:(1) Functional and transcriptomic analysis of the regulation of osteoblasts byMGF-Ct24E.①The roles of MGF-Ct24E in proliferation, differentiation, and mineralization ofrat calvarial osteoblasts were investigated. And the results showed that MGF-Ct24E promotes proliferation while delaying terminal differentiation of osteoblasts in prophase,conceivably to allow the accumulation of more cells to prepare for differentiation andmineralization in more advanced stages of bone formation.②Microarray was carried to determine the molecular mechanisms of boneformation regulated by MGF-Ct24E. The result showed that2054genes in osteoblstswere regulated. Among them,1179genes were found up-regulated while875geneswere down-regulated. Quantitative real-time PCR experiment results showed that theexpression levels of selected twelve differentially expressed genes (IL1RN, FIGF,CXCL12, ZW10, NOTHCH2, TWIST2, ALPL, IGFBF5, FGFR2, COMP, COL2A1andIGFI) were generally in accordance with the microarray data, validating the reliabilityof microarray experiment results.③The GoSurfer software and online software DAVID were applied to GO andKEGG pathway analysis of the screened differentially expressed genes in osteoblastsregulated by MGF-Ct24E. GO analysis of the2054differential expressed genes inbiological process，molecular function and cellular component, the results showed thatthe main enrichment of functional classification were cell cycle process, enzymebinding and intracellular part. KEGG pathway analysis results showed that67pathwayswere modulated by MGF-Ct24E, such as focal adhesion, cell cycle, regulation of actincytoskeleton, gap junction, MAPK signaling pathway, adheres junction, and insulinsignaling pathway, and so on.④Based on GO analysis,95osteogenesis-related genes were screened out from the2054differential expressed genes in osteoblasts. The biological process of GO analysisresults showed that the main enrichment of functional classification of them were asfollowing: cell proliferating, cell cycle, bone development, angiogenesis, regulation ofossification, cartilage ossification, bone mineralization, ossification and so on. KEGGpathway analysis results showed that nine pathways associated with focal adhesion,regulation of actin cytoskeleton, ECM-receptor interaction, cytokine-cytokine receptorinteraction, MAPK signaling pathway, adherens junction, TGF-beta signaling pathway,leukocyte transendothelial migrationk, Wnt signaling pathway and so on weremodulated by MGF-Ct24E.(2) Functional and transcriptomic analysis of the regulation of osteoclasts byMGF-Ct24E.①The roles of MGF-Ct24E in the proliferation, differentiation, and mineralizationof osteoclasts were investigated. The results show that MGF-Ct24E not only inhibits the proliferation, but also inhibits the formation and differentiation of osteoclasts inconcentration-dependent manner. In addition, MGF-Ct24E also shows a significantinhibitory effect on bone absorbing.②Microarray was carried to determine the molecular mechanisms of boneabsorbing regulated by MGF-Ct24E. The result shows that1712genes in osteoclastswere regulated. Among them, about717genes were found up-regulated while895genes were down-regulated. Quantitative real-time PCR experiment results showed thatthe expression levels of selected eight differentially expressed genes (AKT2, SPP1, NF1,ITGB5, NFATc1, TRAF6, MMP9and CTSK) were generally in accordance with themicroarray data, validating the reliability of microarray experiment results.③The GoSurfer software and online software DAVID were applied to GO andKEGG pathway analysis of the screened differentially expressed genes in osteoclastsregulated by MGF-Ct24E. GO analysis of the1712differential expressed genes inbiological process，molecular function and cellular component, the results showed thatthe main enrichment of functional classification were cell cycle process, enzymebinding and intracellular part. KEGG pathway analysis results showed that20pathwayswere modulated by MGF-Ct24E, such as cell cycle，DNA replication，mismatch repair,nucleotide excision repair, homologous recombination, TGF-beta signaling pathway,p53signaling pathway, VEGF signaling pathway, and so on.④Based on GO analysis,79osteoclastogenesis-related genes were screened outfrom the1712differential expressed genes in osteoclasts. The biological process of GOanalysis results showed that the main enrichment of functional classification of themwere as following: cell differentiation, organ development, bone development, bonemineralization, bone remodeling, osteoblast differentiation, developmental process,multicellular organismal development, system development, regulation of ossification,osteoclast differentiation, bone development and so on. KEGG pathway analysis resultsshowed that ten pathways associated with focal adhesion, cytokine-cytokine receptorinteraction, MAPK signaling pathway, VEGF signaling pathway，regulation of actincytoskeleton, leukocyte transendothelial migration, chemokine signaling pathway, tightjunction, TGF-beta signaling pathway, Wnt signaling pathway and so on weremodulated by MGF-Ct24E.In summary, all the results suggest that MGF-Ct24E has a remarkable ability toincrease bone formation and inhibit bone resorption through regulating genes involvedin cell cycle process and six signaling pathways, such as focal adhesion, MAPK signaling pathway, regulation of actin cytoskeleton, TGF-beta signaling pathway,leukocyte transendothelial migration, Wnt signaling pathway and so on.