BMP7 Signaling Via BMPR1A And BMPR1B Regulate The Proliferation Of Pulmonary Large Carcinoma Cell NCI-H460

IntroductionBone morphogenetic protein 7(BMP7) is a signaling molecule originally identified based on its ability to form bone.It is essential during development,and more recently has also been implicated in cancer pathogenesis.Alarmo EL recently demonstrated that BMP7 can promote and inhibit cell growth in breast cancer cell lines. More interestingly,they find growth changes were due to distinct mechanisms since BMP7 silencing led to growth inhibition via G1 arrest in BT-474 cells,whereas BMP7 treatment protected MDA-MB-231 cells from apoptosis.BMP recepters are surface serine/threonine kinase receptors which consist two types.There are three typeⅠreceptors(BMPR1A,BMPR1B,ACVR1)and three typeⅡreceptors.TypeⅠreceptors are the most important parts of the signaling network.So we propose that different functional roles for BMP7 in cancer may due to different typeⅠreceptors concerned in cancer cells.Here,we examined the expression of BMP7 and BMP typeⅠreceptors (BMPⅠR) in human Non-small cell lung tumor cell lines(NSCLC) and Normal human bronchial epithelial cell(HBE) as well as the responsiveness to recombinant human BMP7 in pulmonary large carcinoma cell(NCI-H460).In the last,we investigated the mechanicism of BMP7 signaling by treat NCI-H460 with BMP7and ant-BMP typeⅠreceptors(BMPⅠR).Materials and Methods1.Cell and reagentsHBE(Normal human bronchial epithelial cell),A549(pulmonary adenocarcinoma cell),pulmonary NCI-H460(large carcinoma cell),anti-bmp7,anti-bmpr1a, anti-bmpr-1b,anti-ACVR1A(Santa Cruz),Human recombinant BMP7 protein(R&D Systems),RT-PCR Kit(Takara).2.Cell cultureThe human lung cancer cell line used in this study was routinely grown in RPMI1640,supplemented with 10%fetal bovine serum(FBS),100U/ml penicillin and 100 U/ml streptomycin at 37℃in a5%CO2 incubator.3.Western blotProtein concentration was measured by Bradford method.Cell lysates were electrophoresed in 12%polyacrylamide SDS gel and transferred onto polyvinylidene difluride membranes,Protein bands were blocked and incubated with the first (dilution:bmp7/bmpr1a/bmpr1b/ACVR1A:1:1:200) and second antibody and visualized with DAB kit.Protein contents were calculated by densitometry.4.Reverse Transcription-Polymerase Chain Reaction(RT-PCR)Total RNA was isolated from cells in the logarithmic growth phase using RTISOL (Invitrogen).Follow the instruction of RT-PCR Kit(TaKaRa).5.Proliferation assays1×103 cells were seeded in a 96-well plate,incubated for 24 to 120 h,the MTT and dimethyl sulfoxide(DMSO) were added according to the protocol of the manufacturer.The OD value of each well was measured using a microplate reader with a test wavelength of 490 nm.6.Flow cytometryFlow cytometry were perfermed using Annexin V-FITC Apoptosis Detection Kit I according to the manufacturer's protocol on BD FACSCalibur^TM Flow Cytometer.7.Matrigel invasion assays100μl Cells were added to the upper chamber of an 8μm pore size coated with Matrigel.600μl RPMI1640/10%FBS was added to the lower chamber and cells were allowed to migrate for 24 hours.Cells were fixed in 4%Paraformaldehyde and stained with hematoxylin.The migrated cells were counted in 20 random high-power fields.8.Statistical analysisThe SPSS 14.0 software was employed to analyze the data.P＜0.05 was considered as statistical significance.Results1.The mRNA and protein expression of BMP7 in three NSCLC and HBE cell linesThe HBE cell has the highest level of BMP7 mRNA and protein.All there pulmonary cancer cell lines express relatively low levels of BMP7 mRNA and protein., especially the NCI-H460 cell line.2.The mRNA and protein expression of BMP typeⅠreceptors in three NSCLC and HBE cell lines.BMPR1A is expressed in all of the three NSCLC and HBE cell lines.BMPR1B is expressed in three NSCLC cell lines but not HBE cell line.ACVR1A is expressed in NCI-H460 cell line only.3.The consequences of BMP7 treatment on NCI-H460 cell proliferationBMP7 treatment results in reduced proliferation of NCI-H460 cell.4.The consequences of BMP7 treatment on NCI-H460 cell proliferation after blocking endogenous BMPR1A,BMPR1B and ACVR1A respectivelyBlocking endogenous BMPR1A,BMPR1B obviously reversed the inhibration of BMP7 on NCI-H460 cell proliferation respectively.More over,blocking both endogenous BMPR1A and BMPR1B almost offset the effect of BMP7 on NCI-H460 cell proliferation completely.But ACVR1A blocking did not affect NCI-H460 cell proliferation.5.The effect of BMP7 treatment on apoptosis of NCI-H460 cellsThere was no difference between BMP7 and vehicle treatment with regard to the number of apoptotic cells(data not shown).6.The effect of BMP7 treatment on migration and invasion of NCI-H460 cellsThe total area of migrated cells dcreased 61.7%after treated by BMP7 compared to vehicle-treated cells.Moreover,when NCI-H460 cell were subjected to the invasion assay through Matrigel basement membrane matrix,the total area of invaded cells after BMP7 treatment was 75%less than compared tovehicle treatment.In conclusion, BMP7 treatment significantly decrease NCI-H460 cell migration and invasion.Conclusion1.BMP7 inhibit proliferation,migration and invasion of NCI-H460 cell line.2.BMP7 Signals via BMPR1A and BMPR1B regulate the proliferation of pulmonary large carcinoma cell NCI-H460.