Inhibition Of BMP Signaling And Heterotopic Ossification By AMPK

Chapter 1 IntroductionHeterotopic ossification(HO)is the pathologic formation of ectopic bone in soft tissues.This process usually occurs in two cases: complications with severe trauma,including large surface-area burns,musculoskeletal injury,hip replacement,and even spinal cord injury;and fibrodysplasia ossificans progressiva(FOP),a rare genetic disease.Active mutations of activin receptor like kinase 2(ALK2),one of BMP type I receptors,have been found in almost all FOP patients.The clinical sequela of these pathologic ectopic bone formations,include nonhealing wounds,chronic pain,and joint immobility.Patients die of restriction of respiration due to systemic and massive heterotopic ossification.Mounting evidence has demonstrated that bone morphogenetic proteins(BMPs)and the cognate signaling pathway play an important role in HO.BMPs have been shown to be the primary inducers of HO;Injury and inflammation induces secretion of BMPs in the injured soft tissues,leading to activation of BMP signaling and thereby HO.Likewise,the active mutation of ALK2,majority of which bore R206 H result in constitutive activation of BMP signaling pathway.FOP is characterized by progressive development of symptoms of massive heterotopic ossifications in soft tissues.HO becomes a common and difficult health issue.There is no effective clinical management except mitigation of symptoms by pain relief,rehabilitation,and prophylaxis.The latter relies mostly on anti-inflammatory drugs which is at best only partially effective.Also,side effects are the barrier for widespread use of NSAIDs.An ideal treatment strategy would be to uniquely target components of the BMP signaling pathway and inhibits HO.Alternatively,a different but equally innovative approach would be to identify clinically available drugs used to treat other diseases and re-task this therapeutics to fulfill this clinical need.Previous literature indicates AMPK plays an important role in bone metabolism.Metformin is the first line medication for type 2 diabetes that has been safely used for more than half a century.We have found that AMPK inhibits TGF-beta signaling and EMT.Then I extended the study by investigating the effect of AMPK on BMP signaling pathway and heterotopic ossification.I first investigated the inhibitory effect of AMPK on BMP signaling pathway and osteogenesis of iPS cells derived from FOP fibroblast cells and to study the possible mechanism;To ascertain if the findings reflected different cell context,I also tested the inhibition of AMPK on BMP signaling pathway in MC3T3-E1 cells and the effect of different AMPK activators(single use or combination)on osteogenic differentiation of the cells.Totest if they exhibit dose dependent manner and cooperative effect.Finally,Established animal models and tested the efficacy of AMPK activator in prevention of HO in vivo.Altogether;the study provides a novel view on the mechanism underlying AMPK regulation of BMP signaling pathway and HO.It also provides us a novel insight by developing AMPK activator such as metfomin into treatment of FOP,an incurable disease and HO.Chapter 2 AMPK inhibits BMP signaling pathway in FOP fibroblast cellsObjective: To examine if AMPK activation inhibits the BMP signaling pathway and explore the mechanism underlying inhibition of BMP signaling by AMPK.Methods: To confirm heterozygous mutation of ALK2(R206H)in FOP fibroblast cells by DNA sequencing analysis;FOP fibroblast cells were treated with different AMPK activators,Western blot was used to test the effect of AMPK on key components of BMP signaling pathway;To ascertain if metformin is mediated by AMPK by expressing a constitutive active mutant or dominant negative mutant of AMPKa1 into FOP cells.To construct LKB1 and AMPK a1 a2 stable knockdown in MEF cells and assess the effect metfomin on the response of these cells to BMP6;To establish Firefly Luciferase complementation assay and assess if AMPK activation leads to regulate the interaction between Smurf1 and Smad6,ALK2 and Smad1;si RNA for Smad6 and Smurf1 were transfected into FOP cells and the cells were incubated with MG132 to explore the mechanism underlying inhibition of BMP signaling by AMPK.Results: Sequencing analysis confirmed the heterozygous mutation of ALK2(R206H)in FOP fibroblast cells;Different AMPK activators including metformin and aspirin inhibited Smad1/5 phosphorylation;Smad1/5 phosphorylation was also suppressed by expressing an active mutant of AMPK into FOP cells.The effect of metformin was diminished by infected adenovirus encoding the dominant negative mutant of AMPK into FOP cells;Metformin diminished BMP6-induced phosphorylation of Smad1/5 in MEF wild type cells,which was abolished by knock down of LKB1 or AMPK a1a2 in MEF cells.Activation of AMPK induces degradation of ALK2 and upregulation of Smad6 and Smurf1 expression;the interaction between Smad6 and Smurf1 was enhanced by AMPK activation,while the interaction between ALK2 and Smad1 was markedly diminished.Knock down of Smad6 or Smurf1 blocked the inhibitory effect of AMPK on BMP signaling pathway and prevented metformin induced reduction of ALK2.Furthermore,we also found that MG132,a proteosome inhibitor,blunted the degradation of ALK2 by AMPK.Conclusions:These results suggest that AMPK activated by a variety of maneuvers including pharmacological activators and mutagenesis inhibit Smad1/5 phosphorylation.AMPK activation inhibits BMP signaling pathway in FOP fibroblast cells.The inhibitory effect of AMPK was mediated by upregulation of Smad6 and Smurf1,which leads to increase the interaction of these two molecules and subsequentdegradation of ALK2.Chapter 3 AMPK inhibits osteogenic differentiation of iPS cells derived from FOP fibroblast cellsObjective:To study the effect of AMPK activation on osteogenic differentiation of iPS cells derived from FOP fibroblast cells.Methods:To generate iPS cells derived from FOP fibroblast cells with episomal plasmid vectors and iPS colonies were isolated,amplified and characterized.iPS cells were induced to differentiate into osteogenic cells.Alizain red s staining and Western blot were used to test the osteogenic differentiation ability of iPS cells with ALK2 WT(Control iPS cells)or ALK2 R206H(FOP iPS cells).To test the effect of AMPK activators including metformin and AICAR on mineralization of iPS cells by using Alizarin red s staining.Results: The iPS cells derived from FOP fibroblast cells were successful induction and isolation.FOP iPS showed a trend toward mineralization by using Alizarin red s staining.Western blot showed that the protein level of osteogenic markers including Runx2,OPN and Osc werehigher in FOP iPS than in control iPS after differentiation.AMPK activators metformin and AICAR greatly diminished mineralization of FOP and control iPS cells.Conclusions:The results showed that the iPS cells with ALK2 R206H(FOP iPS cells)exhibited better differentiation than those with wild type ALK2(Control iPS cells).AMPK inhibits osteogenic differentiation of iPS cells derived from FOP fibroblast cells.Chapter 4 The inhibition of AMPK on BMP signaling pathway in mouse pre-osteoblast MC3T3-E1 cellsObjective: To investigate the effect of AMPK on the BMP signaling pathway and explore the mechanism underlying inhibition of BMP signaling by AMPK in MC3T3-E1 cells.Methods:Mouse pre-osteoblast cells MC3T3-E1 were treated with AMPK activator metformin for different times or at varying concentration,Western blot was used to test the effect of AMPK on key components of BMP signaling pathway,cell extracts were blotted with ALK2,Smad6 and Smurf1;Adenovirus encoding the active mutant of AMPK was infected into cells to study the directly effect of AMPK on the BMP signaling pathway;si RNA for Smad6 was transfected into MC3T3-E1 cells to explore the mechanism underlying inhibition of BMP signaling by AMPK.Results:We manipulated AMPK activation using metformin and active mutant of AMPK adenovirus,AMPK activation inhibited BMP6-induced Smad1/5 phosphorylation in MC3T3-E1 cells;Activation of AMPK did not cause decrease in ALK2 and Smurf1,but it increased Smad6 expression;Knock down of Smad6 prevented the inhibitory effect of AMPK on BMP signaling pathway.Conclusions:AMPK activation inhibits BMP signaling pathway in MC3T3-E1 cells.The inhibitory effect of AMPK on BMP signaling pathway was mediated by up regulation of Smad6.Smad6 is the major target for AMPK in inhibiting BMP signaling in MC3T3-E1 cells.Chapter 5 The inhibition of AMPK osteogenic differentiation in MC3T3-E1 cellsObjective: To investigate the effect of AMPK on the osteogenic differentiation in MC3T3-E1 cells.Methods:MC3T3-E1 cells were induced to differentiate into osteogenic cells;Western bolt and q PCR were used to detect the changes in osteogenic differentiation makers and AMPK activity during the differentiation of cells.To study the role of AMPK in the osteoblast differentiation by expressing a constitutive active mutant or dominant negative mutant of AMPKa1 into MC3T3-E1 cells during the differentiation.MC3T3-E1 cells were cultured in osteogenic differentiation medium in the presence of different AMPK activators including metformin,aspirin,curcumin and ibuprofenat varying dose,After one week,The alkaline phosphatase(ALP)staining was performed to test the effect of AMPK on the early event of differentiation;Combine metformin with curcumin or ibuprofen to investigate if they show cooperative effect on the inhibition of ALP activity.Alizarin red s staining was used to assess if AMPK influences mineralization of MC3T3-E1 cells,the later event of osteoblast differentiation.Results:AMPK activity was reduced during the differentiation of MC3T3-E1 cells,AMPK activators including metformin,aspirin,and curcumin and ibuprofrn suppressed the alkaline phosphatase activity in a dose dependent manner.The phosphatase activity was also inhibited by infected with adenovirus encoding the active AMPK mutant,whereas this effect was blocked by the dominant negative mutant of AMPK.Combination of metformin with ibuprofen showed superadditive effect on inhibition of ALP activity,while metformin and curcumin combination displayed additive effect.With increased doses,aspirin and metformin progressively suppressed mineralization of cells.Conclusions:AMPK plays a critical role in osteogenic differentiation.AMPK activation suppressed both the early and late eventof osteogenic differentiation in MC3T3-E1 cells.Metformin and ibuprofen display superadditive effect on the inhibition of ALP activity maybe through different mechanism,whilemetformin and curcurmin show additive effectmay act through a similar mechanism.Chapter 6 The efficacy of AMPK in prevention of heterotopic ossification formation in vivoObjective: To investigate the effect of AMPK on the heterotopic ossification formation in vivo.Methods: To establish mouse trauma/burn model of heterotopic ossification by receiving Achilles tenotomy combined with burn injury.Mice were subsequently treated with 0.5mg/ml metformin in drinking water.After 8 weeks,the injured limbs were examined by x-ray to test the effect of metformin on the HO formation in mice.The HO tissues were fixed in 10% neutral buffered formalin for 24 h and subsequently decalcified in 10%(vol/vol)Nitric acid solution for 3 days.H&E staining was performed to identify the HO structure and cell composition;The Alcian blue staining was performed to test the effect of AMPK on the chondrogenesis.q PCR was used to detect the m RNA expression of osteogenic markers(Runx2,BSP,OSX)and Smad6,Smurf1 after received metformin treatment.Results: We have made a reliable model of HO formation in mice through the use of an Achilles tenotomy combined with a distant burn injury.The injured limbs were examined by X-RAY at 8 weeks after injury.HO developed around calcaneus and ankle joint of the limb that received the tenotomy.The tenotomy mice treated with metformin found a reduction in HO formation at 8 weeks after injury.H&E staining showed that heterotopic ossification tissues in control mice contained abundant cartilage,chondrocytes and osteocytes,No cartilage structure was formed in metformin treated group.Alcian blue staining showed that Alcian blue-positive cartilaginous nodules were formed in control group,few if any formed in metformin-treated samples;Osteogenic marker genes,including the early osteogenic gene Runx2 and the later event makers gene BSP and Osc were decreased in the metformin treated group,while the level of Smad6 and Smurf1 was significantly upregulated.Conclusions: Trauma/burn is a reliable model to produce heterotopic ossification in mice.Metformin prevents trauma/burn induced heterotopic ossification in vivo.