Nitric oxide balances osteoblast and adipocyte lineage differentiation via the JNK/MAPK signaling pathway in periodontal ligament stem cells

Critical tissues that undergo regeneration in periodontal tissue are of mesenchymal origin; thus, investigating the regulatory mechanisms underlying the fate of periodontal ligament stem cells could be beneficial for application in periodontal tissue regeneration. Nitric oxide (NO) regulates many biological processes in developing embryos and adult stem cells. The present study was designed to investigate the effects of NO on the function of human periodontal ligament stem cells (PDLSCs) as well as to elucidate the underlying molecular mechanisms.

NO is essential for maintaining the balance between osteoblasts and adipocytes in PDLSCs via the JNK/MAPK signaling pathway. NO balances osteoblast and adipocyte lineage differentiation via JNK/MAPK signaling pathway.

Immunofluorescent staining and flow cytometry were used for stem cell identification. Western blot, reverse transcription polymerase chain reaction (RT-PCR), immunofluorescent staining, and flow cytometry were used to examine the expression of NO-synthesizing enzymes. The proliferative capacity of PDLSCs was determined by EdU assays. The osteogenic potential of PDLSCs was tested using alkaline phosphatase (ALP) staining, Alizarin Red staining, and calcium concentration detection. Oil Red O staining was used to analyze the adipogenic ability. Western blot, RT-PCR, and staining were used to examine the signaling pathway.

Human PDLSCs expressed both inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) and produced NO. Blocking the generation of NO with the NOS inhibitor L-NG-monomethyl arginine (L-NMMA) had no influence on PDLSC proliferation and apoptosis but significantly attenuated the osteogenic differentiation capacity and stimulated the adipogenic differentiation capacity of PDLSCs. Increasing the physiological level of NO with NO donor sodium nitroprusside (SNP) significantly promoted the osteogenic differentiation capacity but reduced the adipogenic differentiation capacity of PDLSCs. NO balances the osteoblast and adipocyte lineage differentiation in periodontal ligament stem cells via the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) signaling pathway. Conclusions: NO is essential for maintaining the balance between osteoblasts and adipocytes in PDLSCs via the JNK/MAPK signaling pathway. NO balances osteoblast and adipocyte lineage differentiation via JNK/MAPK signaling pathway.