Effects And Mechanisms Of Growth/differentiation Factor-5 On Osteogenic Differentiation In Human Ligamentum Flavum Cells

BackgroudOssification of ligamentum flavum(OLF) is characterized by a heterotopic bone formation in the spinal ligamentum flavum that is normally composed of fibrous tissues.It has been reported more often in Asian populations,particularly in Japanese subjects.The incidence rate is as high as 20%in Asian populations older than 65 years and the ratio of male to female subjects reported in the literature varies about 2-4:1.OLF can be seen in the cervical,thoracic and lumbar region,but it occur significantly more frequently in the lower thoracic region or the thoracic-lumbar junction.The ossified ligament protrudes into the spinal canal and compresses the spinal cord,resulting in serious neurological damages.Therefore,OLF is now appreciated as an important cause of thoracic myeloradiculopathy.Although en bloc laminectomy has been commonly used for treating the condition,the outcome of surgical treatment of thoracic OLF is not always satisfactory.Moreover,the delays of diagnosis,multilevel ossification and postoperative recurrence of ossification in the adjacent segment have a negative effect on functional recovery of the compressed spinal cord.There has been growing concern regarding OLF over the past two decades.Although numerous studies showed that systemic and local factors, including the genetic factors,mechanical stress,growth factors,cytokines,and the endocrine/metabolic abnormalities,were responsible for the pathogenesis of OLK its precise pathogenesis has not been conclusively established.Histological studies in the OLF samples demonstrated that the ossific ligamentum flavum showed loss of elastic fibers and increase of collagen fibers,and the numerous fibrocartilaginous cells were observed within and around the ossification fronts,which supported this theory that the developmental mode of OLF was endochondral ossification.Immunohistochemical studies have also documented that osteogenesis cytokines,such as bone morphogenetic proteins(BMPs), transforming growth factor(TGF)-β,and the receptors of BMP(BMPRs) were localized around the ossification front.These causative factors could play a role in initiation of cellular events that ligamentum flavum(HLF) cells differentiate into chondrocytes and osteoblasts,which further supported by the subsequent studies.In our previous study,the OLF cells,which were isolated from the ligamentum flavum tissues from OLF patients during surgery,showed phenotypic characterization of osteoblasts.In the past decade,several in vitro studies showed that some factors,such as mechanical stress and leptin,induces osteogenic differentiation in HLF cells. Therefore,the osteogenic differentiation of HLF cells may be is a key event in the genesis and development of OLF.Mitogen-activated protein kinases(MAPK),including a variety of isoenzyme, are a family of serine/threonine kinases that play an essential role in signal transduction by modulating gene transcription in the nucleus in response to changes in the cellular environment..The ERK1/2,p38 and JNK of them were found earlier, and their biological functions were extensively investigated.They regulate diverse processes ranging from proliferation and differentiation to apoptosis.Increasing evidence showed that the MAPK pathways are involved in the proliferation and differentiation of osteoblast.It was recently demonstrated that mechanical stress and leptin which are known as the cause of OLF,induced osteogenic differentiation in HLF cells through the activation of MAPK pathways.Thus,the activation of MAPK pathways may be associated with the genesis and development of OLF.Growth/differentiation factor-5(GDF-5),known as BMP-14 or cartilage-derived morphogenetic protein-1(CDMP-1),is a new member of the TGF-beta superfamily, and is also a subfamily of the highly conserved group of bone morphogenetic protein (BMP) signaling molecules.GDF-5 gene,a 488 kb fragment,locates on chromosome 20q11.2,and consists of only two coding exons and one intron.It encodes polypeptide with a total of 501 amino acids,consists of the N-terminus signal peptides,the precursor peptide and C-terminal mature peptides.The post-translational precursor protein split into mature peptides,which play a role in biological activity. The molecular weight of mature GDF-5 dimer is of 25KD,and the monomer molecules weight is of 13.6KD.GDF-5 is known to play crucial roles in skeletal, tendon and ligament morphogenesis.Like other members of the TGF-beta superfamily,GDF-5 transduces its effects through binding to serine/threonine kinase receptors,then activates Smad-dependent and Smad-independent signaling and modulates gene transcription in the nucleus. Recently,some researchers have considerable concerns on the effect of MAPK signaling pathway in the biological activation of GDF-5.in vitro studies have showed that GDF-5 could induce the phosphorylation of ERK in human umbilical vein smooth muscle cells,and induce the phosphorylation of ERK and p38 MAPK in a mouse chondrogenic cell line,ATDC5.The in vivo studies have been demonstrated that GDF-5 promotes the closure of osteochondral defects in a minipig model by enchondral ossification,and induces posterolateral lumbar fusion in a New Zealand white rabbit model.Other studies also showed that the matrices loaded with rhGDF-5 induced ectopic cartilaginous and osseous tissue when implanted in subcutaneous or intramuscular site of rats.Several studies have been demonstrated that it could induce in vitro the osteogenic differentiation in many cell types,such as mesenchymal cell C2C12,fat-derived stromal cells,marrow mesenchymal stem cells and periosteum-derived cells.Histopathological study found that GDF-5 was detected in spindle-shaped cells and chondrocytes in the OLF tissues,but no in the cells in non-ossified sites.The results suggested that GDF-5 may be involved in the progression of OLF.However,it precise pathogenesis is still unclear.Therefore,the identification of the molecular mechanisms of GDF-5-induced OLF has implications for clarifying further the pathogenesis of OLF,and may offer some new information in the prevention,early intervention and treatment of this disease.Objectives1.To explore the method of the isolation and culture of HLF cells in vitro,and establish this cell lines.2.To investigate whether GDF-5 induces osteogenic differentiation in HLF cells.3.To investigate the effect of GDF-5 on the phosphorylation of MAPKs,and investigate the effect of MAPK inhibitors on GDF-5-induced osteogenic differentiation in HLF cells.Methods1.Isolation and culture of HLF cells in vitro.Tissue samples of ligamentum flavum were harvested during surgery from patients with thoracolumbar burst fractures or from patients with lumbar disc herniation.The samples were washed with phosphate-buffered saline(PBS),after which any surrounding tissues attached to the specimen were carefully removed.The collected ligaments were minced into pieces of approximately 0.5 mm³ and washed twice with PBS.The minced tissue was digested at 37℃for 90 min with 0.2% collagenase type I in serumless containing DMEM.Collagenase-treated ligament chips were washed with DMEM and then placed in 6-well plates or 10 ml dishes in DMEM supplemented with 10%fetal bovine serum(FBS),100 U/ml penicillin and 100 pg/ml streptomycin,and incubated in a humidified atmosphere of 95%air and 5%CO₂ at 37℃.The medium was firstly changed at day 5-7 after explant culture and then replaced at three-day intervals.The outgrown cells grown to confluence were subcultured using trypsinization with 0.2%trypsin/0.02%ethylenediamine tetraacetic acid(EDTA).Under an inverted phase microscope,the cultures were examined for cell outgrowth and Cell morphology and growth status.MTT colorimetric method was used to analysis the proliferation of HLF cells at the first,third and fifth passage.Cell growth curve was then drawed and cell population doubling time was calculated.We compared the biological characteristics of the HLF cells within 5 generation. Immunofluorescence staining was used to detect the expression of vimentin and collagen type I.2.To investigate whether GDF-5 induces osteogenic differentiation in HLF cells(1) Alkaline phosphatase(ALP) activity assayThe OLF cells were exposed to 100ng/mlrhGDF-5 for 0,4,7,10,14 days or 0,10, 50,100,500ng/ml rhGDF-5 for 10 days,respectively.Cells were collected and their total protein was then prepared with RIPA lysis buffer.The ALP activity was examined using the commercial reagents.(2) Determination of the expression of osteocalcin mRNA in HLF cellsThe OLF cells were exposed to 100ng/mlrhGDF-5 for 0,4,7,10,14 days or 0, 10,50,100,500ng/ml rhGDF-5 for 10 days,respectively.Cells were collected and Total RNA was extracted using the TRI Reagent ^? according to the manufacturer's instruction.The expression of osteocalcin mRNA was analyzed by reverse transcriptase-polymerase chain reaction(RT-PCR).Relative expression of osteocalcin was calculated usingβ-actin mRNA expression as an internal control.(3) Determination of the expression of osteocalcin protein in HLF cellsThe OLF cells were exposed to 100ng/ml rhGDF-5 for 0,4,7,10,14 days or 0, 10,50,100,500ng/ml rhGDF-5 for 10 days,respectively.Cells were collected and their total protein was then prepared with RIPA lysis buffer.The expression of osteocalcin protein was analyzed by Western blot.(4) Determination of the location expression of osteocalcin protein in HLF cellsHLF cells were planted on glass coverslips in 6-well plates,and cultured in the DMEM medium containing 10.0%FBS.Cells grown to 80%confluence were treated with or without 100ng/mlrhGDF-5 for 10 days.Immunofluorescence staining was used to detect the location expression of osteocalcin.The coverslips were treated by 1:100 dilution of goat polyclonal anti-osteocalcin antibody and subsequent 1:50 dilution of FITC fluorescent-labeled goat,and observed under the confocal microscope.(5) Alizarin red stainingHLF cells were planted in 6-well plates,and cultured in the DMEM medium containing 10.0%FBS.Cells at confluence were treated with or without 100ng/ml rhGDF-5 for 28 days.Mineralized nodule formation was examined by the alizarin red staining.3.The signal transduction mechanism of rhGDF-5-induced osteogenic differentiation in HLF cells.(1) Determination of the phosphorylation of MAPKsThe OLF cells were exposed to 100ng/ml rhGDF-5 for 0,5,15,30,60,120,240 min,and collected for protein isolation.Inhibition experiments were performed by 1 h pretreatment with the MAPKs inhibitors U0126,SB203580 and SP600125 before rhGDF-5 stimulation.The total protein and phosphorylated protein of MAPKs (ERK1/2,p38 and JNK) was analyzed by Western blot.(2)Effect of MAPKs inhibitors on rhGDF-5-induced osteogenic differentiation in HLF cells.The OLF cells were exposed to 100ng/ml rhGDF-5 for 10 days with or without 20μM U0126,SB203580 and SP600125,respectively.The ALP activity and osteocalcin protein were examined.Results1.Isolation and culture of HLF cells in vitro.(1) Inverted phase microscopeHLF cells were successfully isolated using a collagenase-pretreated explant method.Within 14 days after explants,outgrowth of cells was observed from ligament tissue explants and became monolayer.Morphologically,HLF cell lines varied widely in appearance,and ranged from thin,spindle-shaped cells to polygonal cells or oval-shaped cells.The primary cells showed radial growth at sub-confluence. The passage cells arranged mainly spindle-shaped,some were fan-shaped or polygonal.They showed vortex-like growth at sub-confluence.When HLF cells subculture continuously to the fifth passage,they still maintain a good morphology.(2) The proliferation,growth curve and cell population doubling time in the passage cells.HLF cells were planted at a density of 2×10⁴ cells per well in 96-well plates. They grow rapidly and showed S-shaped growth curve,0-3 days for the incubation phase of cell growth;3-5 days for proliferative phase;thereafter cell growth entering the plateau.In the MTT analyses,the proliferation of the P1,P3 and P5 cells were no significant difference(F＝0.188,P＝0.831).The cell population doubling times were no significant difference(F＝0.405,P＝0.675).(3) Immunofluorescence stainingImmunofluorescence staining showed that the vimentin and collagen type I expressed in the all HLF cells.2.rhGDF-5 induces osteogenic differentiation in HLF cells.(1) rhGDF-5 induces increase of ALP activity in HLF cellsAfter the stimulation of HLF cells with 100ng/ml rhGDF-5 for 0,4,7,10,14 days or 0,10,50,100,500ng/ml rhGDF-5 for 10 days,rhGDF-5 induces increase of ALP activity in a time- dependent(F＝273.905,P＝0.02) and dose -dependent manner(F＝64.091,P＝0.004).(2) rhGDF-5 induces expression of osteocalcin mRNA in HLF cellsRT-PCR measurement showed that rhGDF-5 treatment resulted in a time-dependent(F＝46.573,P＝0.018) and dose-dependent(F＝70.300,P＝0.002) increasing expression of osteocalcin mRNA.(3) rhGDF-5 induces expression of osteocalcin protein in HLF cellsWestern blot measurement showed that rhGDF-5 treatment resulted in a time-dependent(F＝287.754,P＝0.001) and dose-dependent(F＝115.087, P＝0.004) increasing expression of osteocalcin protein.Immunofluorescence staining showed that the expression of osteocalcin was located in the cytoplasmic in the rhGDF-5 group cells,but not in the control group cells.(4) rhGDF-5 induces mineralized nodule formation in HLF cellsAlizarin red staining showed that the mineralized nodule formation was seen in rhGDF-5 treated cells,but not in the control cells.3.rhGDF-5 stimulated the phosphorylation of ERK1/2 and p38 MAPK,their inhibitors U0126 and SB203580 inhibited the rhGDF-5-induced osteogenic differentiation in HLF cells.After the stimulation of HLF cells with 100ng/ml rhGDF-5 for 0,5,15,30, 60,120,240min,Western blot analyses showed that rhGDF-5 treatment resulted in increasing the phosphorylation of ERK1/2 and p38 MAPK in time-dependent manner (F＝75.940,P＝0.008;F＝19.537,P＝0.030),but not affected the the phosphorylation of JNK(F＝0.250,P＝0.688).The pretreatment of ERK1/2 inhibitor U0126 and p38 inhibitor SB203580 could inhibit the phosphorylation of ERK1/2 and p38, respectively.ERK1/2 inhibitor U0126 and p38 inhibitor SB203580 could inhibit the increasing ALP activity and osteocalcin protein induced by rhGDF-5.Conclusions1.The HLF cells can be successfully isolate by collagenase-predigested explant culture.HLF cells in primary culture showed fibroblast-like phenotype.It is stabile that the biological characteristics of cells within 5 generations2.rhGDF-5 can in vitro induce osteogenic differentiation in HLF cells through the ERK1/2 and p38MAPK pathways.