title: Growth/differentiation factor-5 induces osteogenic differentiation of human ligamentum flavum cells through activation of ERK1/2 and p38 MAPK.
abstract: Ossification of ligamentum flavum (OLF) is a pathological ectopic ossification in the spinal ligament, leading to spinal canal stenosis, but little was known about its pathogenesis. A previous study has found growth/differentiation factor (GDF)-5 expression at ossified sites of the ligaments from OLF patients. This study aimed to investigate the osteogenic effects of GDF-5 on cultured human ligamentum flavum cells (LFCs). LFCs were isolated from human spinal ligamentum flavum, and treated with or without recombinant human (rh) GDF-5. Alkaline phosphatase (ALP) activity was measured. Expression of osteocalcin was assessed by reverse transcriptase-PCR, Western blotting and immunofluorescence. Matrix mineralization was assessed by alizarin red staining. Activation of mitogen-activated protein kinases (MAPK) ERK1/2, p38 and JNK were detected by Western blotting. We found that rhGDF-5 treatment increased ALP activity and osteocalcin expression in a time- and dose-dependent manner, and induced mineralized nodule form. In addition, rhGDF-5 challenge mediated the ERK1/2 and p38 activation but not JNK. Inhibiting this activation pharmacologically, using U0126, a ERK1/2 inhibitor, or SB203580, a p38 inhibitor, resulted in significantly lower ALP activity and osteocalcin protein expression. The present study shows that rhGDF-5 induces osteogenic differentiation of human LFCs through activation of ERK1/2 and p38 MAPK. These findings give some new insight into the pathogenesis of OLF.
authors.affiliation: Department of Orthopedic and Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.
authors.forename: Zhao-Ming
authors.initials: ZM
authors.lastname: Zhong
authors.forename: Jian-Ting
authors.initials: JT
authors.lastname: Chen
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Ding-Sheng
authors.initials: DS
authors.lastname: Zha
authors.forename: Zhuo-Sheng
authors.initials: ZS
authors.lastname: Lin
authors.forename: Cheng-Yi
authors.initials: CY
authors.lastname: Zhao
authors.forename: Jun-Chang
authors.initials: JC
authors.lastname: Xu
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Zi-Xing
authors.initials: ZX
authors.lastname: Xu
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D002070:Butadienes / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D055428:Growth Differentiation Factor 5 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D017843:Ligamentum Flavum / Q000166:cytology / Q000201:enzymology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis; D011725:Pyridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D002070:Butadienes; D055428:Growth Differentiation Factor 5; D007093:Imidazoles; D009570:Nitriles; D011725:Pyridines; D011994:Recombinant Proteins; C113580:U 0126; D015675:Osteocalcin; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D000469:Alkaline Phosphatase; C093642:SB 203580