Nitric oxide modulates osteoblastic differentiation with heme oxygenase-1 via the mitogen activated protein kinase and nuclear factor-kappaB pathways in human periodontal ligament cells.
Nitric oxide (NO) and heme oxygenase-1 (HO-1) play important roles in the regulation of stem cell proliferation and differentiation. However, it has not been examined whether human periodontal ligament (PDL) cells can differentiate into osteoblast-like cells by NO activity mediated via HO-1. The objective of this study was to determine the effect of NO on proliferation and differentiation in human PDL cells, and to identify the underlying mechanism of its actions. Primary human PDL cells were cultured with NO donor sodium nitroprusside (SNP); cell proliferation and differentiation were measured. NO production, cell viability and cell proliferation were evaluated using the Griess reagent, MTT assay and BrdU incorporation, respectively. To analyze differentiation, we measured alkaline phosphatase (ALP) activity, osteocalcin (OC), osteonectin (ON) expression, and bone sialoprotein (BSP) by Western blotting. SNP-induced NO production is associated with inducible nitric oxide synthase induction in a time and dose-dependent manner. SNP resulted in decreased cell proliferation and increased expression of osteogenic differentiation markers such as ALP, OC, ON and BSP. Maximal HO-1 was reached with 0.05 mM SNP and gradually decreased with 1.0 mM. Treatment with an HO-1 inhibitor and selective inhibitors of extracellular regulated kinase 1/2 and nuclear factor-kappaB blocked the SNP-induced growth inhibition, as well as osteoblastic differentiation. These data suggest that NO-induced osteogenic differentiation through HO-1 may be an important mediator of periodontal regeneration or bone tissue engineering.
Lee
Sun-Kyung
SK
Department of Oral & Maxillofacial Pathology, College of Dentistry, Wonkwang University, Iksan, South Korea.
Choi
Hee-In
HI
Yang
Yun-Seok
YS
Jeong
Gil-Saeng
GS
Hwang
Joo-Hee
JH
Lee
Sang-Im
SI
Kang
Kyung-Hwa
KH
Cho
Jin-Hyung
JH
Chae
Jong-Moon
JM
Lee
Suk-Keun
SK
Kim
Youn-Chul
YC
Kim
Eun-Cheol
EC
D000469:Alkaline Phosphatase / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051547:Heme Oxygenase-1 / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis; D009599:Nitroprusside / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects; D010513:Periodontal Ligament / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D010766:Phosphorylation
D004791:Enzyme Inhibitors; D016328:NF-kappa B; D020030:Nitric Oxide Donors; D009599:Nitroprusside; D009569:Nitric Oxide; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D051547:Heme Oxygenase-1; D020928:Mitogen-Activated Protein Kinases; D000469:Alkaline Phosphatase