| Background and ObjectiveOsteogenesis imperfecta(OI),also known as brittle bone disease,is a group of genetically heterogeneous connective tissue disorders characterized by disturbances in collagen metabolism,skeletal dysplasia,increased bone fragility,spontaneous fractures,and impaired bone healing.Currently,drug therapy is the mainstay of treatment for OI.The majority of the drugs used to improve skeletal dysplasia and bone abnormalities in OI were originally developed for the treatment of osteoporosis,but they still have serious side effects and complications that should not be overlooked.At present,the drugs used to treat OI are primarily focused on inhibiting osteoclastosis.Although drugs that promote osteogenesis are used to treat OI,they have not fundamentally corrected the abnormal bone formation.There are still no effective drugs that target the pathological mechanism of OI.Therefore,there is a strong incentive to investigate the pathogenesis of OI and find new therapeutic targets,which will be of great significance for the prevention,diagnosis and treatment of OI.Type Ⅰ collagen is the main component of bone extracellular matrix,accounting for approximately 90%of bone organic matrix,which is critical for preserving bone integrity and maintaining skeletal function.The abnormal structure or quantity of type Ⅰ collagen in OI causes abnormal bone metabolism,which is characterized by attenuated osteoblast differentiation,decreased osteogenic mineralization,and an increase in the number of undifferentiated osteoblasts.The undifferentiated osteoblast secretes large amounts of RANKL,which causes osteoclast formation and increased bone resorption activity,resulting in osteopenia,fractures,and bone abnormalities in OI patients.Therefore,promoting osteoblast differentiation and maturation is crucial for essentially correcting the imbalance of bone metabolism in OI.Based on the abnormal type Ⅰ collagen,in-depth exploration of the underlying mechanism of abnormal osteogenic differentiation in OI will help to find novel intervention targets and therapeutic strategies for OI.Transforming growth factor-β(TGF-β)is a class of multifunctional cell regulatory proteins found in bone matrix that play an essential role in bone metabolism.Excessive TGFβ/Smad signaling is one of the major causes of abnormal osteogenic differentiation in OI,but the precise process by which type Ⅰ collagen deficiency leads to increased TGF-β/Smad signaling remains unknown.The intracellular transport of TGF-β receptor(TβR)is an important way to regulate TGF-β/Smad signaling and is primarily mediated by Caveolindependent endocytosis and Clathrin-dependent endocytosis.Caveolin-dependent endocytosis-mediated TβRs intracellular transport degrades TβRs(primarily TβRⅠ),reducing TGF-β/Smad signaling,whereas Clathrin-dependent endocytosis-mediated TβRs intracellular transport activates the Smad2/3 signaling pathway,increasing TGF-β/Smad signaling.The role of these two endocytosis pathways in modulating TGF-β/Smad signaling in OI pathogenesis remains to be elucidated.The interaction of type Ⅰ collagen with integrinα2β1 caused a reduction in the cell surface expression of TβRs without affecting mRNA expression in osteoblastic cells.Meanwhile,the downregulation of TβRs by type Ⅰ collagen may be involved in FAK activation and its downstream signal transduction.Caveolin-1(Cav-1),a principal protein of caveolae,was initially identified for substrates of Src kinase.Src-dependent phosphorylation of Cav-1 plays a crucial role in the regulation of caveolae formation,release,and trafficking.From this,we hypothesize that type Ⅰ collagen regulates Cav-1 phosphorylation-mediated TβRs degradation and TGF-β/Smad signaling transduction by activating integrin α2β1/FAK/Src signaling pathway.In summary,we put forward a hypothesis:The integrin a2β1/FAK/Src signaling pathway cannot be effectively activated due to the deficiency of type Ⅰ collagen in OI,resulting in a decrease in Cav-1 phosphorylation and impairment of the Cav-1 phosphorylation-dependent endocytic pathway.Which further affects TβRⅠ degradation and increases TGF-β/Smad signaling,ultimately leading to abnormal osteogenic differentiation of OI.In order to verify this hypothesis,we chose an OI mouce model(B6C3fe-α/αColla2oim mouse)as the object and performed molecular biology,cell biology,and in-vivo animal experiments on it.Firstly,we confirmed that type Ⅰ collagen can reduce the sensitivity of osteoblasts to TGF-β stimulation.Secondly,we further verified that type Ⅰ collagen can mediate TβRα degradation via the caveolin-dependent endocytic pathway,and an in-depth investigation was conducted on the exact mechanism of how type Ⅰ collagen regulates Cav1 phosphorylation.Finally,the in vivo therapeutic effect of inhibiting TβRα kinase on OI was investigated.This study is expected to uncover a novel mechanism of abnormal osteogenesis in OI,and provide new targets and ideas for the treatment of OI.Part Ⅰ:type Ⅰ Collagen induced TβRⅠ degradation and negatively regulated TGF-β/Smad signaling.Methods:(1)Obtain osteoblasts derived from OI mouse model oim/oim mice and wildtype wt/wt mice as research objects.Quantitative real-time PCR(qRT-PCR),ALP staining and Alizarin Red S(ARS)staining were used for the detection of osteogenic differentiation and mineralization.(2)After TGF-β intervention,qRT-PCR and western blot were used to assess osteogenic differentiation and the activation of Smad signaling.(3)Western blot was used to detect the expression and degradation of TβRs on the membrane surface of oim/oim osteoblasts and wt/wt osteoblasts.type Ⅰ collagen was extracted from wt/wt or oim/oim mice tails and incubated with osteoblasts before being analyzed by western blot for the degradation of TβRs.Results:(1)qRT-PCR results showed that the osteogenic differentiation of oim/oim osteoblasts was dramatically impaired,and the expression levels of osteogenic marker genes Runx2,ALP,and OCN were significantly reduced.ALP staining and ARS staining demonstrated that osteogenic differentiation into the mature stage was inhibited in oim/oim osteoblasts.(2)After TGF-β intervention,qRT-PCR and western blot revealed that the osteogenic marker genes Runx2,ALP,and OCN were considerably lowered in oim/oim osteoblasts,while the phosphorylation levels of Smad2 and Smad3 were obviously higher in oim/oim osteoblasts than wt/wt osteoblasts.(3)Western blot indicated that the protein levels of TβRⅠ and TβRⅡ on the membrane surface of oim/oim osteoblasts were significantly higher than in wt/wt osteoblasts,and the TβRⅠ and TβRⅡ degradation of oim/oim osteoblasts was hampered.Normal type Ⅰ collagen could induce downregulation of T(3Rs(TβRⅠ and TβRⅡ)on the cell membrane surface,whereas defective type Ⅰ collagen in OI fail to induce downregulation of TβRs.Conclusions:When compared to wt/wt osteoblasts,oim/oim osteoblasts exhibited impaired osteogenic differentiation and were more sensitive to the osteogenic inhibitory effect of TGF-β.The expression of TβRs(TβRⅠ and Tβ)on the membrane surface of oim/oim osteoblasts was significantly higher than that of wt/wt osteoblasts,which may be related to TβRs degradation caused by defective type Ⅰ collagen.Part Ⅱ:The role of Caveolin-1-dependent endocytic pathway in type Ⅰcollagen-mediated TβRⅠ degradation.Methods:(1)Extraction of membrane protein samples from oim/oim osteoblasts and wt/wt osteoblasts,lipid raft and nonlipid raft fractions were further separated and identified.(2)Immunofluorescence staining and western blot were used for detecting the distribution of TβRⅠ degradation pathway in oim/oim osteoblasts and wt/wt osteoblasts.(3)The caveolae/raft-mediated endocytosis was preblocked,or pretreatment osteoblasts with lysosome inhibitor/proteasome inhibitor,then osteoblasts were incubated with normal typeⅠ collagen and western blot was used for detecting endocytic degradation of TβRⅠ.Co-IP assay was used for assessing TβRⅠ ubiquitination after normal type Ⅰ collagen intervention.(4)The expression level of Cav-1 and the phosphorylation level of Cav-1 in oim/oim osteoblasts and wt/wt osteoblasts were detected by western blot.Oim/oim osteoblasts and wt/wt osteoblasts were incubated with type Ⅰ collagen extracted from wt/wt or oim/oim mice tails,and western blot was used for evaluating Cav-1 expression and the phosphorylation of Cav-1.After transfecting osteoblasts with a control vector or a dominant-negative Cav-1 Y14(Tyr14)plasmid and incubating them with normal type I collagen,western blotting was used to assess Cav-1 phosphorylation and TβRⅠ degradation.Osteoblasts pretreated with anti-integrin α2β1 antibody and incubated with normal type Ⅰ collagen,then Cav-1 phosphorylation and TβRⅠ degradation were evaluated by western blot.(5)Following pretreatment with FAK inhibitor or Src kinase inhibitor,oim/oim osteoblasts and wt/wt osteoblasts were incubated with type Ⅰ collagen,and Cav-1 phosphorylation and TβRⅠdegradation were measured using a western blot.Results:(1)In oim/oim osteoblasts,the distribution of TβRⅠ was decreased in lipid raft fractions while increased in nonlipid raft fractions.(2)In oim/oim osteoblasts,the Caveolaemediated TβRⅠ degradation pathway was attenuated,while the Clathrin-mediated endocytic pathway was enhanced.(3)Western blot demonstrated that pretreatment with MβCD or genistein blocked the downregulation effect of type I collagen on TβRⅠ.The degradation of TβRⅠ by type I collagen in osteoblasts was reversed by lysosome inhibitors NH4Cl and chloroquine,or proteasome inhibitor MG 132.(4)Western blot indicated that treatment with integrin α2β1 neutralizing antibody decreased the level of Cav-1(Tyr14)phosphorylation and markedly reversed the reduced levels of TβRⅠ induced by type Ⅰ collagen.(5)Inhibition of FAK or Src kinase significantly decreased the level of Cav-1 phosphorylation and greatly reversed type Ⅰ collagen-induced TβRⅠ downregulationConclusions:In oim/oim osteoblasts,the distribution of TβRⅠ was decreased in lipid raft fractions and the Caveolae-mediated TβRI degradation pathway was attenuated.Normal type Ⅰ collagen could promote TβRⅠ ubiquitination and induced its degradation,which is mediated by regulating Cav-1 phosphorylation through integrin α2β1/FAK/Src signaling pathway.Defective type Ⅰ collagen in OI dysregulates integrin α2β1/FAK/Src-mediated Cav1 phosphorylation and TβRⅠ degradation,resulting in enhanced TGF-β/Smad signaling.Part Ⅲ:Therapeutic effect of the inhibition of TβRⅠ kinase on OI mouse model.Methrods:(1)Four-week-old male oim/oim and wt/wt mice were randomly divided into four groups based on genotype(Oim or Wt)and treatment(Vehicle or SD-208):Oim/Vehicle group,Oim/SD-208 group group,Wt/Vehicle group and Wt/SD-208 group.TβRⅠ kinase inhibitor SD-208(60 mg/kg/day)or 1%methylcellulose(Vehicle)was fed by oral gavage for 8 consecutive weeks.(2)Before treatment(4-weeks old)and every 2 weeks during the treatment period,whole body X-ray was performed to capture the occurrence of fractures until the end(12-weeks old).(3)After the experiment,isolated left femurs were scanned by micro-CT for bone microarchitecture analysis.(4)Right femurs were evaluated for the measurements of bone biomechanical characteristics.(5)Left tibias were harvested for dynamic bone histomorphometry.(6)Right tibias were obtained for immunohistochemical staining.Results:(1)Inhibition of TβRⅠ kinase decreased fracture incidence in oim/oim mice.(2)Inhibition of TβRⅠ kinase improved bone microstructure of oim/oim mice.(3)The result of three-point bending test suggested that inhibition of TβRⅠ kinase improved the bone biomechanical properties of oim/oim mice.(4)The results of dynamic bone formation indicated that inhibition of TβRⅠ kinase enhanced bone formation and bone mineral apposition in oim/oim mice.(5)Immunohistochemical staining revealed that inhibition of TβRⅠ kinase could effectively correct the imbalance of bone metabolism in oim/oim mice,which was manifested by stimulating osteogenesis and inhibiting osteoclast formation.Conclusions:TβRI kinase inhibitor SD-208 could effectively promote bone formation and inhibit osteoclast formation,positively improve bone microstructure and bone biomechanical properties,thereby reducing the occurrence of fractures in oim/oim mice. |
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