Strain Reduction of Human Gingival Fibroblasts Induces the ATP Pathway

Abstract

Studies in rats demonstrated that surgical detachment of marginal gingiva from root surfaces induced alveolar bone resorption via activation of ATP receptor P2X4. Our aim was to study the effects of strain release of human gingival fibroblasts by detachment of collagen coat from culture dishes. Changes in cell shape, extracellular ATP, cell calcium and genes relevant to trigger alveolar bone resorption were measured. HGF cells from human marginal gingiva explants were seeded on collagen coated culture dishes. In the test group, the collagen coat was detached from culture dish 4-5 days after seeding. Quantification of extracellular ATP was measured by Bioluminescent Assay Kit and changes of intracellular calcium by FLU-AM fluorescence. Real-time PCR was used to examine expression of purinergic receptor P2X4 and P2X7, RANK-L and STC1. Cell length of HGFs grown on plastic surface and on collagen was similar. Reduction in cell length of 45% was measured after detachment of collagen coat. Extracellular ATP raised 10 folds 1 minute after detachment of collagen coat, then declined and returned to control levels 60 minutes later. Ionomycin increased extracellular ATP, while, pretreatment by EGTA, or BAPTA-AM, 30 minutes prior to Ionomycin, reduced significantly extracellular ATP. Reciprocally, addition of ATP increased influx of calcium into HGFs. At the molecular level elevated expression of STC-1, RANK-L and P2X7 was recorded by RT -PCR in detached HGFs, while expression of P2X4 was unchanged. Expression of STC-1, a modulator of cellular calcium and phosphate, P2X7 a calcium ionic channel purinergic receptor and RANK-L a powerful regulator of osteoclastic activity, all were up-regulated significantly. Conclusion: We propose that drop of strain of HGF cells and change in their shape stimulated release of cellular ATP which is signaling through Pi/Ca modulators the molecular activation of osteoclasts.