ClC-3 chloride channel mediates the role of parathyroid hormone [1-34] on osteogenic differentiation of osteoblasts

• Published in: PloS one 2017-04, Vol.12 (4), p.e0176196-e0176196
• Main Authors: Lu, Xiaolin, Ding, Yin, Niu, Qiannan, Xuan, Shijie, Yang, Yan, Jin, Yulong, Wang, Huan
• Format: Article
• Language: English
• Subjects: Acidification; Alkaline phosphatase; Alkaline Phosphatase - genetics; Alkaline Phosphatase - metabolism; American Type Culture Collection; Animals; Anthraquinone; Apoptosis; Attention; Biochemistry; Biology and life sciences; Bisphosphonates; Bone density; Bone growth; Bone marrow; Bone mass; Bone mineral density; Bone morphogenetic proteins; Bone remodeling; Bone strength; Carbon dioxide; Cartilage; Cell differentiation; Cell growth; Cell Line; Cell Proliferation - drug effects; Cell Proliferation - physiology; Chloride; Chloride Channels - genetics; Chloride Channels - metabolism; Chloride ions; Core Binding Factor Alpha 1 Subunit - genetics; Core Binding Factor Alpha 1 Subunit - metabolism; Culture media; Degradation; Differentiation (biology); Engineering; Estrogens; Gene expression; Glands; Hematology; Hippocampus; Hypersensitivity; Immunofluorescence; Inactivation; Injection; Ion channels; Kidneys; Kinases; Kinetics; Laboratories; Medicine and Health Sciences; Metabolism; Mice; Military; Mineralization; Nodules; Older people; Orthodontics; Osteoblasts; Osteoblasts - drug effects; Osteoblasts - metabolism; Osteocalcin - genetics; Osteocalcin - metabolism; Osteogenesis - drug effects; Osteogenesis - physiology; Osteopontin - genetics; Osteopontin - metabolism; Osteoporosis; Parathyroid Hormone - pharmacology; Physical Sciences; Proteins; Real time; Research and Analysis Methods; RNA, Small Interfering; Side effects; Signal Transduction - drug effects; Sp7 Transcription Factor; Stem cells; Stimulation; Transcription factors; Transcription Factors - genetics; Transcription Factors - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0176196

Different concentrations of parathyroid hormone [1-34] (PTH [1-34]) can have totally opposite effects on osteoblasts. Intermittent stimulation with PTH can significantly increase bone mineral density in vitro, mainly through the protein kinase A (PKA) signaling pathway, which phosphorylates runt-related transcription factor 2 (Runx2). The ClC-3 chloride channel, an important anion channel, can also promote osteogenesis via the Runx2 pathway based on recent studies. The purpose of our study, therefore, is to research whether the ClC-3 chloride channel has an effect on PTH osteodifferentiation in MC3T3-E1 cells. A cell counting kit (CCK-8) and real-time PCR were used to investigate the impact of different PTH stimulation modes on MC3T3-E1 cell proliferation and osteogenesis-related gene expression, respectively. We found that the minimum inhibitory concentration of PTH was 10-9 M, and the expression of alkaline phosphatase (Alpl) and Runx2 were at the highest levels when treated with 10-9 M PTH. Next, we used real-time PCR and immunofluorescence technique to detect changes in ClC-3 in MC3T3-E1 cells under PTH treatment. The results showed higher expression of the ClC-3 chloride channel at 10-9 M intermittent PTH administration than in the other groups. Finally, we used the ClC-3 siRNA technique to examine the role of the ClC-3 chloride channel in the effect of PTH on the osteogenesis of osteoblasts, and we found an obvious decrease in the expression of bone sialoprotein (Ibsp), osteocalcin (Bglap), osterix (Sp7), Alpl and Runx2, the formation of mineralization nodules as well. From the above data, we conclude that the expression of ClC-3 chloride channels in osteoblasts helps them respond to PTH stimulation, which mediates osteogenic differentiation.