Involvement of GSK3/β‐catenin in the action of extracellular ATP on differentiation of primary cultures from rat calvaria into osteoblasts

• Published in: Journal of cellular biochemistry 2018-11, Vol.119 (10), p.8378-8388
• Main Authors: Laiuppa, Juan A., Santillán, Graciela E.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - pharmacology; Alkaline Phosphatase - metabolism; Animals; ATP; beta Catenin - metabolism; Biocompatibility; Bone growth; Calcification, Physiologic - drug effects; Calvaria; Catenin; Cell culture; Cell differentiation; Cell Differentiation - drug effects; Cell Survival - drug effects; Cells, Cultured; Differentiation (biology); Extracellular matrix; Glycogen Synthase Kinase 3 - antagonists & inhibitors; Glycogen Synthase Kinase 3 - metabolism; GSK3; Inhibition; Lithium chloride; Lithium Chloride - pharmacology; Mineralization; Nuclei (cytology); osteoblast differentiation; Osteoblastogenesis; Osteoblasts; Osteoblasts - metabolism; Osteogenesis; Osteogenesis - drug effects; Protein Translocation Systems; Purine receptors; purinergic receptors; Rats; Receptors; Signal Transduction - drug effects; Skull - cytology; Translocation; Uridine Triphosphate; βcatenin
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.27037

Modulation of purinergic receptors play an important role in the regulation of osteoblasts differentiation and bone formation. In this study, we investigated the involvement of the GSK3/βcatenin signaling in the action of ATPγ‐S on osteogenic differentiation of primary cell cultures from rat calvaria. Our results indicate that the cell treatment with 10 or 100 µM ATPγ‐S for 96 h increase the cytoplasmic levels of β‐catenin and its translocation to nucleus respect to control. A similar effect was observed after cell treatment with the GSK3 inhibitor LiCl (10 mM). Cell treatments with 4‐10 mM LiCl significantly stimulated ALP activity respect to control at 4 and 7 days, suggesting that inhibition of GSK‐3 mediates osteoblastic differentiation of rat calvarial cells. Effects comparison between ATP and LiCl shown that ALP activity was significantly increased by 10 µM ATPγ‐S and decreased by 10 mM LiCl at 10 day of treatment, respect to control, suggesting that the effect of ATPγ‐S was less potent but more persistent than of LiCl in stimulating this osteogenic marker in calvarial cells. Cell culture mineralization was significantly increased by treatment with 10 µM ATPγ‐S and decreased by 10 mM LiCl, respect to control. In together, these results suggest that GSK3 inhibition is involved in ATPγ‐S action on rat calvarial cell differentiation into osteoblasts at early steadies. In addition such inhibition by LiCl appear promote osteoblasts differentiation at beginning but has a deleterious effect on its function at later steadies as the extracellular matrix mineralization. ATPγ‐S stimulates citoplasmic accumulation of β‐catenin and its nuclear translocation in primary cell cultures from rat calvaria. The similar effect on β‐catenin nuclear translocation induced by cell treatment with 10 mM LiCl or 10 and 100 μM ATPγ‐S, suggest the involvement of GSK‐3 inhibition in the purinergic signaling on osteoblasts. At short time (4‐7 days) LiCl strongly increased alkaline phosphatase activity, whereas ATPγ‐S do it more gently at day 10 of treatment. Long time treatment (15‐22 days) with ATPγ‐S increase, whereas with LiCl decrease culture mineralization. In addition LiCl also decrease cell viability from day 4 on later, suggesting that if well at beginning seems improves osteoblasts differentiation, later has a deleterious effect.