Growth/differentiation factor-5 induces osteogenic differentiation of human ligamentum flavum cells through activation of ERK1/2 and p38 MAPK.
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.
Zhong
Zhao-Ming
ZM
Department of Orthopedic and Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Chen
Jian-Ting
JT
Zhang
Yu
Y
Zha
Ding-Sheng
DS
Lin
Zhuo-Sheng
ZS
Zhao
Cheng-Yi
CY
Xu
Jun-Chang
JC
Li
Tao
T
Xu
Zi-Xing
ZX
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*
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